50 years of Galactolipid Research: The Beginnings

Introduction

This review assumes that the basic facts of galactolipid biosynthesis are known, and instead of presenting another review, it tries to recall how research on this topic started more than 50 years ago and then rapidly spread into many other areas of plant lipid biochemistry. Galactolipids came into focus of plant lipid research in the late 1950s as by-products of investigations on photosynthetic 14CO2 fixation in green algae. Even before their final structural elucidation in 1961, their identification induced a change in plant lipid research by expanding the prevailing interest concentrated on oilseed fatty acids to metabolism and function of membrane lipids. The continuously important position of research on galactolipids is documented by the fact that from their very beginning in 1974, all international meetings on plant lipid biochemistry had central sessions on glycolipids [1].

Following their discovery and structural elucidation, it took just about two decades to recognize their important implications in different sections of plant lipid physiology. On the other hand, satisfactory answers to many questions raised in the beginning could not be provided due to experimental limitations. This left plant lipids for many years as a field for specialists with admittedly important, but not precisely understood correlations with the other areas of plant research. Nevertheless, an ever growing community of plant lipidologists accumulated an extended knowledge on many details of lipid structures and metabolism. This rapidly paid off once the methodologies of molecular biology and genetics had been established and enabled targeted interference studies which finally led plant lipids out of their ghetto. In the following, an attempt is made to trace back the origin of some of the important ideas with lasting relevance for galactolipid research. Due to the limited space, relatively few efforts can be covered.

 

Structural Identification and Quantification

The two galactolipids monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG) were discovered in wheat flour and analyzed in Carter’s laboratory with substantial contributions by his two Ph.D. students E.D. Slifer and C. Tipton. Except for the localization of fatty acids and the stereochemistry at C2 of the glycerol part, all details of the crystalline deacylated compounds were published in 1956 [2]. NMR and MS methods were not used in these studies, but a first GLC profile identified linoleic acid as the predominant fatty acid [3]. The structural studies were completed in 1961 by a final methylation analysis of the intact galactolipids demonstrating that the two acyl groups were linked to the glycerol part thus forming a diacylglycerol moiety [4]. The stereochemistry of this moiety as sn-1,2-diacylglycerol (in stereospecific nomenclature) was proposed by comparison with natural and synthetic reference compounds which had become available from Wickberg’s studies on osmoprotectants of red algae in 1958 [5,6].

The first structural investigations on MGDG and DGDG from leaves, including the diagnostic data of the deacylated and crystallized backbones, were reported by Sastry and Kates in 1964 [7]. In a different approach for the stereochemical assignment of the glycerol moiety, the primary hydroxyl group of the glycerol part of the deacylated glycolipid was oxidized to a carboxyl group and the subsequently released glyceric acid identified by crystallization as the L-isomer [8]. In the intact glycolipids, this stereoisomer corresponds to an sn-1,2-diacylglycerol moiety. This work, carried out in Benson’s lab after radiolabelling experiments with Chlorella, was submitted just half a year later than Carter’s final paper [4], suggesting that parallel efforts were going on in the two labs. From acknowledgments regarding the exchange of crystalline reference compounds, it is evident that there was some cooperation between the labs of Carter, Wickberg and Benson. For the sulfolipid, the assignment of the glycerol stereochemistry was confirmed by X-ray analysis of the deacylated and crystallized sulfoquinovosylglycerol [9]. A recent confirmation of the stereochemistry for the three chloroplast glycolipids was based on chemical release of the diacylglycerol part from the glycolipids followed by derivatization and chiral HPLC resulting in a clear separation of enantiomeric diacylglycerols [10].

Neither in the “Introduction” nor in the “Discussion” of any of their three papers [2-4] did the discoverers of the galactolipids point out the reasons why they went through the efforts to fractionate a lipid extract from 50 kg of wheat flour as provided by Procter & Gamble. Therefore, one is tempted to assume that at the beginning of their work, Carter’s group did not expect to retrieve glycolipids of this structure which later turned out to represent the major lipid constituents of plants. The earth’s most abundant lipids were apparently discovered by chance without realizing their importance.

In contrast, from work on photosynthetic fixation using CO2 labelling experiments in Chlorella, Benson’s lab was well aware of the importance of these glycolipids. As early as 1958, Benson pointed out that the galactolipids, isolated two years ago in Carter’s lab, represent important membrane lipids of photosynthetically active organisms and outnumber phospholipids by a factor of four as deduced from labelling studies using Carter’s crystalline compounds as references [11]. In the same paper, they also reported the labelling of trigalactosyldiacylglycerol, TGDG, and suggested that galactolipid biosynthesis may require uridine diphosphate-galactose.

During this time (1958) Wintermans was a postdoctoral fellow in Benson’s lab. Inspired by these developments and after returning to the Netherlands, he published the first quantitative analysis of glyco- and phosphoglycerolipids in leaves and chloroplasts in 1960 [12], i.e. already one year before the final structures of the two galactolipids were released. Thin-layer chromatography of leaf lipids was not in use yet, but was introduced a few years later in 1963 [13]. Wintermans based his quantification on separation of deacylated lipids by two-dimensional paper chromatography developed in Benson’s lab using Carter’s reference compounds. According to Wintermans’ data, the two galactolipids were exclusively localized in chloroplasts and in fact were the predominant lipids in leaves. He missed the occasion to label MGDG as “the most abundant polar lipid in nature” and to obtain a citation record as did his successors in this issue [14].

 

Galactolipids and Photosynthesis

Shortly later a more comprehensive “lipidomics” approach described the proportions of the predominating lipophilic components in thylakoid membranes including chlorophylls, carotenoids, plastoquinones, tocopherols and glycerolipids by compiling data from different laboratories [15]. In combination with electron micrographs showing crystalline arrays in the thylakoid surface (published as cover on a Science issue), the same data were used to calculate the molecular composition of the “quantasome” [16]. This entity is difficult to ascribe to a presently known substructure, but the two papers [15,16] were placed in Nature and Science for a broad dissemination of the new developments in plant science. Many years later, maize thylakoids were used to directly quantify the same set of lipophilic components with roughly similar results [17]. Even for modern lipidomics techniques, the parallel quantification of all these compounds, referenced to sets of calibration standards, is a challenge [18]. With the establishment of TLC separation and GLC of fatty acids, the quantification of plant galacto- and phospholipids became an easy task. The formerly very small plant lipidology community grew, many physiological issues could be approached and the publication number on plant glycolipids increased. Up to this point, the limited number of publications allowed a chronological description of the progress. But because of the subsequent parallel efforts in many different directions, this will be given up in the following to focus on a few selected issues.

The repeatedly observed parallel increase of galactolipids and chlorophyll during greening of cells and tissues confirmed the assumption that close structural and functional correlations exist between thylakoid lipids and photosynthesis as proposed by Benson [11]. Right from the beginning, one of the great challenges was a detailed understanding of molecular interactions between the different components forming the thylakoid membrane. In the early 1960s, this field was discussed in reviews by Benson [19] and Menke [20] who had shortly before coined the term “thylakoid” and investigated its dimensions by wide-angle diffraction X-ray studies. Based on his studies with Euglena, a widely used organism for studies on fatty acid biosynthesis [21], Rosenberg suggested a molecular model in Science. It emphasized the hydrophobic association between four unsaturated acyl groups of galactolipids and one phytol residue of chlorophyll with a constant ratio in the lipid bilayer [22].

It took many years and required different experimental approaches to resolve this problem and to realize that chlorophylls and other pigments are protein-bound. In fact, all the hydrophobic thylakoid components mentioned above [15-17], including glyco- and phospholipids, were detected in different electrophoretically separated protein complexes [23]. This was shown by numerous immunological studies with lipid-specific antisera prepared by Radunz in Menke’s laboratory, but never summarized in a review. These results were substantiated by quantitative lipid analyses of pure complexes isolated on a larger scale [24]. The functional relevance of galactolipids for photosynthesis was later confirmed by genetic studies, as mutants of Arabidopsis deficient in MGDG [25,26] or DGDG synthesis [27] clearly demonstrated an in vivo role of these two lipids for photosynthetic efficiency.

But only recently, after the resolution of X-ray structures of crystallized protein complexes isolated from thylakoid membranes had dropped below 3 Å, did the molecular details of protein, lipid and pigment interactions become visible in great detail [28-30]. Unexpectedly, all structural parts of the resolved galactolipids, and in particular their acyl groups, contribute to the formation of pockets to bind the chlorin ring of chlorophylls rather than interacting with its phytol residue [29]. The identification of the lipids in different locations of photosystems I and II suggests many new functions and places them into the position of new cofactors not only involved in the modulation of exciton/electron transport [28-30], but also of de novo synthesis of the various subunits and of repair exchange processes [24]. The proportion and functional relevance of the (galacto)lipids that are not engaged in protein interactions and that occur free in the bilayer of thylakoids still remain unsolved. It will be interesting to see whether galactolipids are engaged in similar interactions in the other membrane systems in which they are found.

 

(Galacto)Lipid-linked Desaturation

After the discovery of galactolipids, a wide interest was directed towards their fatty acid components. Before the common use of TLC for separation of plant lipids, the determination of fatty acids in lipids required their isolation by column chromatography making such analyses rare. In 1960 Weenink [31] submitted the first analysis of the fatty acid profile of MGDG isolated from leaves (clover), which contained nearly pure (95.8%) linolenic acid, C18:3. A similarly high percentage (95.5%) was found in MGDG from runner-bean leaves [7], whereas Allen et al. reported for the first time the high proportion (30%) of hexadecatrienoic acid, C16:3, in MGDG from spinach leaves [32]. In subsequent studies, it was confirmed to be the ∆7,10,13-isomer [33] and to be localized to the sn-2-position of MGDG [34]. These studies led to the identification of two plant families (Chenopodiaceae, Fabaceae), which differ by the presence of C16:3 in MGDG. Many years later and based on an increased amount of data from different laboratories [35], such plant families were named 16:3-plants in contrast to the prevailing 18:3-plants [36]. In the meantime, the positional analyses of cyanobacterial lipids had suggested that the sn-2 location of C16:3 in MGDG of eukaryotic plants may be a typically prokaryotic characteristic of chloroplasts [32,34-38] .

The remarkable fatty acid profiles of galactolipids immediately raised questions regarding the desaturation reactions leading to the prevailing C16- and C18-trienoic acids. The status of this field in the early 1960s was covered in a comprehensive review correlating fatty acid patterns with phylogenetic relations between cyanobacteria, single-cell and multicellular algal groups, protists, fungi, higher plants and animals. In this review, Erwin and Bloch tried to reduce the bewildering variety of double bond patterns to a few generalizations [39]. Many of these earlier studies were concerned with the delineation of a “plant pathway” leading from oleic to α-linolenic acid. But important details such as the localization of fatty acids in different lipids as well as their distribution between the sn-1/2-positions had hardly been looked at.

This was changed when James, Nichols, Gurr and coworkers entered the field. This group, not working at a university but in an English Unilever research laboratory, increased the analytical resolution by combining silver ion TLC separation of molecular species with positional analyses of fatty acids in MGDG [40] and phosphatidylcholine (PC) [41,42]. Their studies on the turnover of lipids and their individual fatty acids in Chlorella suggested that desaturation occurred subsequent to the incorporation of acyl groups into polar lipids. Based on these data, the authors developed a new paradigm for the biosynthesis of polyunsaturated fatty acids in plants. They extended the acyl-CoA/acyl carrier protein-thioester-based model by adding lipid-bound acyl groups as new substrates for desaturases. This crucial contribution to general biochemistry was first published in 1967 [43] and confirmed in subsequent studies [34,40-42].

Despite its importance, the actual substrates used by various organisms in different desaturation reactions have only occasionally been identified. The first clear and detailed scheme of MGDG-linked desaturation, published in 1970 for Chlorella, depicted the sequential desaturation of the 18:0/16:0 species to the 18:3/16:2 species [34]. In view of present day knowledge, only the first desaturation step of 18:0 to 18:1 is considered not to be lipid-linked in chloroplasts [44,45], whereas the extended studies by Murata and coworkers showed that in cyanobacteria, both C16:0 and C18:0 acyl groups are desaturated as ester components of polar lipids [46]. In the first experiments with MGDG from leaves of a 16:3 plant, Siebertz and Heinz showed that all desaturation intermediates from 16:0 to 16:3 are exclusively localized at the sn-2 position, suggesting the formation of this double bond series via lipid-linked desaturation [47]. These results were later confirmed by studies with isolated spinach chloroplasts [48]. The crucial C16:0-∆7-desaturase FAD5 of Arabidopsis was cloned many years later [45]. At this point it may be added that not only cyanobacteria [46] and plants [45], but also bacteria [49] contain a desaturase that is capable of desaturating the sn-2-linked saturated acyl chain of lipids.

The Unilever group was always cautious enough to point out that in vivo labelling experiments do not allow the exclusion of alternative pathways, particularly a rapid and tightly coupled exchange of acyl groups between lipid and desaturase. But after opening this new area of lipid biochemistry, the three authors left the field without executing, as announced [41], the conclusive desaturase experiment with lipids carrying ether-linked substrate chains to exclude the exchange mechanism. These desaturation experiments with octadecenyl ether analogous substrates as suggested in 1969 were finally carried out more than 20 years later by Schmidt, Sperling, Heinz and co-workers. After incubation of a solubilized ω6-desaturase with a labelled octadecenyl ether analog of MGDG as substrate [50], reversed phase-HPLC showed the formation of two desaturation products, suggesting desaturation at both the sn-1  and sn-2 positions. Parallel in vitro and in vivo experiments with octadecenyl ether analogs of PC also showed desaturation of sn-1- and sn-2-linked substrate chains [51,52]. Thus, lipid-linked desaturation represents a strategy in plants not questioned any more, but in biotechnological approaches, it can be an obstacle for subsequent elongation reactions of polyunsaturated fatty acids which requires the transfer to the corresponding acyl-CoA-thioesters.

The genes encoding lipid-linked plant desaturases were finally isolated via a forward genetic approach initiated by Somerville and co-workers based on the GLC-based screening for Arabidopsis mutants with altered fatty acid composition [53]. This strategy resulted in the first map-based cloning of a plant gene, FAD3, encoding the PC-linked C18:2 desaturase of the ER [54]. Isolation of FAD3 and of the additional desaturases confirmed the existence of two sets of lipid-linked desaturases operating in the chloroplast and the ER [44]. Only the first desaturation step from stearic acid to oleic acid, is thioester-linked [55,56].

 

Another Surprise

The first detailed acetate-labelling studies of leaf lipids were carried out by Roughan in 1970 [57], at the time, when the work on lipid-linked trienoic acid formation in Chlorella had just been published [34,40-43]. His acetate labelling experiments with expanded leaves of pumpkin, an 18:3 plant, revealed another surprising detail. At the beginning, labelling was particularly high in PC and comparatively low in MGDG, which was reflected in linolenic acid labelling. But in the course of 3 days, there was a shift of label from PC to MGDG. Apart from a confirmation of PC-linked desaturation, Roughan was brave enough to deduce a new hypothesis to account for this additional phenomenon: “If α-linolenic acid is synthesized primarily within the PC molecule then it follows that this fatty acid would be incorporated into other glycerolipids by acyl transfer” [57]. It should be recalled that this conclusion was drawn at a time when membrane flow and fusion, lipid transfer proteins and lipid transporters were not as familiar as they are today. In many subsequent studies in collaboration with Slack and others, this at first provocative hypothesis was corroborated.

To substantiate the subcellular implications, the experimental strategy was extended to studies with isolated chloroplasts and microsomal membranes. Double-labelling experiments with 18:3 plants suggested that not free fatty acids, but 18/18-diacylglycerol backbones were transferred from microsomal PC to chloroplasts for formation of eukaryotic 18/18-MGDG [58]. On the other hand, sequential addition of substrates to chloroplasts isolated from spinach, a 16:3 plant, resulted in the formation of prokaryotic 18/16-MGDG [59,48]. After 10 years of research in New Zealand and many other laboratories (just to name those of Mazliak, Williams and Yamada), Roughan, Slack and coworkers formulated a comprehensive concept to fit all the puzzling data [59]. In 1980, this paper presented for the first time the scheme of the “prokaryotic” and “eukaryotic pathway” in plant membrane lipid synthesis and fatty acid desaturation with particular emphasis on the exchange reactions linking microsomal PC and plastidial MGDG. The general relevance of this concept for plant biology was immediately recognized as evident from the invitation to present an annual review [60]. This two-pathway model has proved to be one of the most fruitful concepts developed in plant lipid biochemistry. It made its way into textbooks and stimulated the imagination regarding lipid dynamics in plant organelle membranes. But only in recent years, Benning’s group has started to successfully work through the protein assemblies required for the synthesis of eukaryotic MGDG and to define the actual lipid transport metabolite [61]. On the other hand, the advantage of retaining the formation of prokaryotic glycolipids in chloroplasts of many plants including Arabidopsis, where nearly equal proportions of pro- and eukaryotic MGDG are found, is still not understood [62,63].

 

Enzymatic Reactions for Galactolipid Synthesis

Many of the results described so far would not have been possible without the parallel success in the characterization and subcellular localization of the enzymatic reactions involved in phospho- and galactolipid biosynthesis. For galactolipids, the first in vitro experiments were carried out in 1964 by Neufeld and Hall with spinach chloroplasts [64]. With the use of radioactive UDP-galactose, they confirmed the full sequence of galactosylation reactions up to a putative tetragalactosyldiacylglycerol (TeGDG) as suggested by Benson in 1958 [11]. Remarkably, 83% of the terminal galactose of radioactive DGDG was not α- but β-linked. In most of the subsequent in vitro studies, this important anomeric detail was not looked at, but in view of the work by van Besouw and Wintermans [65] and Benning’s group [66], the formation of this all-β-DGDG as well as the formation of the corresponding TGDG and TeGDG can now be ascribed to the activity of the processive galactolipid:galactolipid galactosyltransferase (GGGT). This activity was completely unknown in the 1960s.

Ongun and Mudd [67] continued on this line and demonstrated that diacylglycerols as well as MGDG and DGDG were used as acceptors for galactosylation. They suggested that two different enzymes may be responsible for formation of MGDG and DGDG from which the DGDG-forming one was less firmly bound to membranes. Allen et al. had already pointed out that the difference in fatty acid profiles between MGDG and DGDG may be relevant for understanding this galactosylation step [32]. At this time chloroplast envelope preparations, crucial for the localization and characterization of the different galactosyltransferases, were not yet available [68].

A major breakthrough was achieved 10 years later, after Mackender and Leech had isolated the first envelope membrane fraction from purified chloroplasts [69]. Douce, as a postdoctoral fellow in Benson’s lab involved in the development of a similar method [70], used this newly available membrane fraction for studies on galactolipid biosynthesis. In 1974, he observed the highest galactosylation activity measured so far [71]. Most importantly, a comparison of galactosyltransferase activities in envelopes and thylakoids suggested that the assembly of galactolipids is concentrated in the plastid envelope. This unexpected distribution required the transfer of galactolipids from envelopes into the thylakoids. Up to now, the molecular details of this transport are not resolved [61,72]. The same is true for the asymmetric distribution of DGDG in the thylakoid membrane [73], which is also observed in crystalline photosystem II [29,30].

The availability of envelope preparations boosted a series of investigations not only on lipids, but on all aspects of plastid biochemistry. The group in Grenoble with Douce, Joyard and their co-workers first concentrated on the analysis of the envelope lipid composition and enzymatic reactions involved in the biosynthesis of lipids and lipophilic components, which are subsequently transferred to the thylakoid membrane. Over the years an impressive collection of data was built up and recently extended by proteomics analyses, which refined the understanding of plastid biology [74]. It took another 10 years, until a method for the separation of inner and outer envelope membranes from intact chloroplasts was established 1981 in Keegstra’s lab [75], while a slightly modified method was subsequently developed in Douce’s lab [76]. As a consequence, the studies on enzymatic activities, lipids and other components measured before with whole envelopes were repeated with isolated outer and inner membranes [74]. In Arabidopsis, the distribution of galactosyltransferases to the two membranes is unequal, since only MGD1 is localized in the inner envelope, whereas MGD2 and MGD3 as well as DGD1 and DGD2 and GGGT are found in the outer envelope [68,66]. A similarly detailed assignment is missing for an 18:3 plant.

Isolated envelope membranes were a tempting starting material for the isolation of galactosyltransferases involved in galactolipid biosynthesis. The efforts in two labs (Maréchal and Teucher in the labs of Douce and Heinz, respectively, both 1991) to isolate the MGDG synthase from spinach envelopes resulted in a 300-500-fold purification, but both groups assigned a protein with an incorrect molecular weight. Finally, Shimojima, Ohta and co-workers, starting with cucumber microsomes, obtained a protein fraction after a 7400-fold purification suitable for peptide sequencing. Two years later in 1997, they published the first nucleotide sequence for a plant UDP-galactose:diacylglycerol galactosyltransferase (MGDG synthase) [77]. This success and the positional cloning of the DGDG synthase, achieved shortly later by Dörmann, Benning and coworkers [78], are the milestones which opened new avenues in plant galactolipid research [68] regarding regulation and function, made possible for example by transcriptomic and interference studies.

But with the three MGDG and two DGDG synthases cloned, not all enzymes involved in galactolipid biosynthesis had been identified yet. Chloroplasts from a dgd1dgd2 double mutant of Arabidopsis still produced TGDG and TeGDG [79], but none of the two DGDG synthases, when expressed in a suitable host, produced the higher homologues TGDG and TeGDG with all-β-configuration [80]. These oligogalactolipids are found in isolated envelope membranes [71,75] and are formed in the in vitro assays mentioned above [64,67,79] as well as in envelope preparations from radioactive UDP-galactose [71]. Already in 1978, van Besouw and Wintermans had suggested a surprising mechanism for DGDG biosynthesis by the galactolipid:galactolipid galactosyltransferase GGGT [65]. In the first step, this processive enzyme uses MGDG both as acceptor and donor of a galactose residue and thus is independent of UDP-galactose. The MGDG and DGDG synthases are UDP-galactose-requiring glycosyltransferases involved in bulk galactolipid synthesis, whereas the GGGT catalyzes a glycosylhydrolase reaction. In contrast to Neufeld and Hall [64], van Besouw and Wintermans did not check the anomeric linkage of the DGDG formed in their assays which was also never done with TGDG and TeGDG identified by MS in isolated envelopes [81]. Therefore, van Besouw and Wintermans [65] had discovered a new reaction in plant galactolipid metabolism and detected a new enzyme, but incorrectly suggested that it was the enzyme responsible for the formation of the “normal” bulk DGDG.

More than 30 years later Benning’s group characterized this enzyme on a molecular level and demonstrated its processivity. They did not miss to determine the anomeric configuration of the GGGT-dependent DGDG, which turned out to be all-β-DGDG [66]. It was a big surprise that the previously identified Sensitive to Freezing 2-locus of Arabidopsis encodes the GGGT protein which unexpectedly contributes to the freezing tolerance by galactolipid remodeling. But it is not clear why GGGT is activated during envelope isolation or by a block in the import of the 18/18-precursor for eukaryotic MGDG synthesis. On the other hand, the formation of TGDG and TeGDG with the two/three terminal galactose residues in α-configuration (summarized in [82]) is presumably catalyzed by one of the DGDG synthases, as it was shown that the Arabidopsis DGD2 protein produces α-anomeric linkages and is processive [80]. The formation of TGDG in cyanobacteria (identification based only on MS [37]) and in Chlorella [11] has not been studied in detail.

Another surprising aspect of galactolipid function was discovered in 2000, as DGDG, but not MGDG, was retrieved in extraplastidial membranes, with a strong increase during phosphate deprivation [83]. The increase in glycolipid production during phosphate deficiency, which was already known from some bacterial species, provides the means to replace phospholipids, enabling plants to survive on low-phosphate soils. The phosphate-deprivation-dependent transfer of DGDG from the chloroplast envelope, the site of synthesis, to target extraplastidic membranes is not understood mechanistically [68].

Because of its relevance for understanding the phylogenetic diversification after primary endosymbiosis, the deviating mode of MGDG biosynthesis in cyanobacteria will be mentioned as a last point. In 1980, labelling studies showed that in cyanobacteria, monoglucosyldiacylglycerol is formed first and then epimerized to MGDG [84]. The UDP-glucose:diacylglycerol glucosyltransferase was characterized enzymatically [85], but for its cloning, a bioinformatics approach had to be followed since no close sequence homology with the plant MGDG synthases exists [86]. In cyanobacteria, this glucosyltransferase might contribute to temperature tolerance [87] by an increased synthesis of the “heat shock lipid” monoglucosyldiacylglycerol [88].

In contrast to cyanobacteria which harbor a glucosyltransferase for the initial step of galactolipid synthesis, plants, the photosynthetic anaerobic bacterium Chlorobaculum [89] and the apicocomplexan-like endosymbiont Chromera [90] contain UDP-galactose-specific MGDG synthases (see above). Therefore, the origin of the plant MGDG synthase cannot simply be traced back to cyanobacteria [86,89,90]. A comparison of presently available sequences suggests that green and red algae, glaucophytes and plants have abandoned the cyanobacterial glucosyltransferase following a horizontal transfer of a bacterial galactosyltransferase into their nuclear genome [90]. This galactosyltransferase is distantly related with bacterial glycosyltransferases, including the MGlcD synthase from Staphylococcus, and with MurG glycosyltransferases involved in bacterial cell wall synthesis [77,90]. Furthermore, this scenario is compatible with the assumption that the membrane proteins in thylakoids have evolved to interact with galactolipids [29], but not with glucolipids [91]. These vital interactions had to be maintained after establishing the primary endosymbiosis, whereas MGDG synthesis could be simplified by replacing two enzymes by one [90] and taking into account a concomitant loss of a glucolipid-based mechanism of heat shock protection.

An even more complicated situation exists with regard to the DGDG synthases. Green algae and plants contain nucleus-encoded enzymes which are localized in the outer envelope membrane [68]. They are unrelated to the cyanobacterial DGDG synthases [92,93] which are distantly related to glycosyltransferases from α-proteobacteria and to glycosyltransferases of primary metabolism of plants. But their exact phylogenetic origin remains enigmatic [80,90,92,93], and the sugar nucleotide specificity (glucose or galactose) of the expressed enzymes has not yet been determined [92,93]. Surprisingly, sequences related to the cyanobacterial DGDG synthases are found as plastid-encoded enzymes in a few single-cell red algae and a glaucophyte [90,92,93] as could be expected for a situation resulting from an endosymbiosis event.

In summary, the selected examples of this retrospective show that galactolipid research contributed to both an understanding of functional aspects and phylogenetic diversification in plant biology. From the ever increasing number of unexpected involvements, recognized by a new generation of plant lipidologists and not covered here, it is not even an exaggeration to conclude that “galactolipids rule in seed plants” [94].

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