The Lipid Library

MONO- and DIGALACTOSYLDIACYLGLYCEROLS and RELATED LIPIDS FROM PLANTS and MICROORGANISMS

STRUCTURE, OCCURRENCE, BIOSYNTHESIS and ANALYSIS

1.  Mono- and Digalactosyldiacylglycerols from Plants

Monogalactosyldiacylglycerols and digalactosyldiacylglycerols (together with the plant sulfolipid – see below) are the main lipid components of the various membranes of chloroplasts and related organelles, and indeed they are the most abundant lipids in all photosynthetic tissues, including those of higher plants, algae and certain bacteria. In non-photosynthetic tissues of plants, the proportion of glycosyldiacylglycerols is greatly reduced. The predominant structures are 1,2-di-O-acyl-3-O-β-D-galactopyranosyl-sn-glycerol and 1,2-di-O-acyl-3-O-(6'-O-α-D-galactopyranosyl-β-D-galactopyranosyl)-sn-glycerol.

In higher plants, the galactolipids of photosynthetic tissues contain a high proportion of polyunsaturated fatty acids, up to 95% of which can be linolenic acid (18:3(n-3)). In this instance, the most abundant molecular species of mono- and digalactosyldiacylglycerols must have 18:3 at both sn-l and sn-2 positions of the glycerol backbone. Plants such as the pea, which have 18:3 as almost the only fatty acid in the monogalactosyldiacylglycerols, have been termed "18:3 plants". Other species, and the 'model' plant Arabidopsis thaliana is an example, contain appreciable amounts of hexadecatrienoic acid (16:3(n-3)) in the monogalactosyldiacylglycerols, and they are termed "16:3 plants". A further distinctive feature is that this acid is located entirely at the sn-2 position of the glycerol backbone (see Table 1). Palmitic acid tends to be found only in digalactosyldiacylglycerols, usually in small amounts, when the positional distribution appears to depend on species. In non-photosynthetic tissues, such as tubers, roots or seeds, the C_l8 fatty acids are usually more saturated (c.f. the data for wheat flour lipids).

On the basis of these structures, galactolipids are classified into two groups. The first has mainly C₁₈ fatty acids at the sn-l position of the glycerol backbone, and only C₁₆ fatty acids at the sn-2 position, and it is termed a "prokaryotic" structure (as it is characteristic of cyanobacteria - see Table 2 below also). The second class has C₁₆ or C₁₈ fatty acids at the sn-l position but only C₁₈ fatty acids in the sn-2 position, and this is termed a "eukaryotic" structure, as it is present in most glycerolipids, such as the phospholipids, of all eukaryotic cells. The exception is phosphatidylglycerol, which is synthesised in chloroplasts via the prokaryotic pathway only. Some plants contain both eukaryotic and prokaryotic structures in the monogalactosyldiacylglycerols.

The structural differences in the diacylglycerol moiety of galactolipids from algae and higher plants are believed to originate in compartmentalization of the biosynthetic pathways in eukaryotic cells, each compartment having its own distinctive enzymes.

2.  Other Non-acidic Glycosyldiacylglycerols from Plants

The main galactosyldiacylglycerols consist of 1,2-di-O-acyl-3-O-β-D-galactopyranosyl-sn-glycerol and its digalactosyl homologue formed biosynthetically by addition of an α-D-galactopyranosyl residue to C6 of the first galactose unit. However, other homologues occur that are formed by a continuation of this process, i.e. trigalactosyldiacylglycerol and tetragalactosyldiacylglycerol. For example, trigalactosyldiacylglycerols have been found in pumpkins and potatoes, and tri- and tetragalactosyldiacylglycerols in rice bran.

In addition, a second series has been identified consisting of all-beta-linked homologues, i.e. linear (1→6)-linked all-β-galactolipids with two to four galactose units. These have been found in a variety of plant families, algae and some bacteria. A third series has been described from one plant species that is derived from the normal (α→β)-linked digalactosyldiacylglycerol by sequential addition of 6-O-β-D-galactopyranosyl residues, resulting in alternative types of tri- and tetragalactosyldiacylglycerol. An interesting poly-glycosylglycerolipid has been found in mung beans with a terminal rhamnose unit and unusually an alkyl group in position sn-2 (in animal glycerolipids, the ether moiety is invariably in position sn-1 - see our web pages on Ether lipids). Oat seeds contain an interesting form of digalactosyldiacylglycerol with an estolide linkage, i.e. 15-hydroxylinoleic acid is esterified to position sn-2, and the hydroxyl group of this is esterified with linoleic acid.

1,2-Di-O-acyl-3-O-β-D-glucopyranosyl-sn-glycerol has been found in rice bran, where it occurs with the corresponding galactolipids in an approximate ratio of 1:2. Interestingly, the two forms differ appreciably in their fatty acid compositions. Triglycosyldiacylglycerols containing a high proportion of glucose have also been found in rice, but the structures have not been confirmed definitively. Although glucosyldiacylglycerols have been found in some other plants, they are always rather minor components.

The phytoplackton Chrysochromulina polylepis contains monogalactosylglycerol linked via the sugar moiety and an ester bond to a chlorophyll pigment.

3.  Biosynthesis and Function of Glycosyldiacylglycerols

The basic biochemical mechanisms of galactolipid synthesis require the synthesis of 1,2-diacyl-sn-glycerols via phosphatidic acid as the first step (as in phospholipid biosynthesis). A monogalactosyldiacylglycerol synthase then effects the reaction of the diacylglycerols with uridine 5-diphosphate(UDP)-galactose to form monogalactosyldiacylglycerols.

A further enzyme system catalyses the addition of another galactose unit from UDP-galactose to form digalactosyldiacylglycerols. In the prokaryotic pathway, which is located in the chloroplast envelope, the phosphatidic acid that is synthesised contains only oleic acid in position sn-1 and palmitic acid in position sn-2. The eukaryotic pathway is located in the endoplasmic reticulum and yields phosphatidic acid with C₁₈ fatty acids in position sn-2 and a C₁₈ or a C₁₆ fatty acid in position sn-1. There is extensive trafficking of diacylglycerols between the various cellular compartments, and the acyl moieties of these are actively desaturated in situ. The final galactolipid structures are governed by the relative activities of the various enzyme systems in different cellular organelles and the rates of exchange between each. The mechanism of this transfer is uncertain but may involve vesicular transport.

There are now known to be three different sets of lipid galactosyltransferases capable of galactoglycerolipid biosynthesis in Arabidopsis thaliana. One set provides the bulk of galactolipids in the chloroplast and photosynthetic tissues in general, while under conditions of phosphate limitation and in non-photosynthetic tissues a second set is highly active. A third pathway does not use UDP-galactose as the donor, but involves a processive enzyme, which transfers galactose from one galactolipid to another. In cyanobacteria, monoglucosyldiacylglycerol is formed first, and this is epimerized to monogalactosyldiacylglycerol.

Because of its small head group, monogalactosyldiacylglycerol has a cone-like geometry with galactose at the point and the two fatty acyl chains oriented towards the base. Therefore, in aqueous systems, it tends to form a hexagonal-II phase, with the polar head group facing towards the centre of micellar structures rather than forming a bilayer. In contrast, digalactosyldiacylglycerols with two galactose moieties in the head group have a more cylindrical shape, so they form lamellar phases and thence bilayers. The ratio of these two lipids must be under tight control for proper membrane function.

It is clear that the galactosyldiacylglycerols have important functions in photosynthesis, and the nature of these functions is the topic of active research although much of the detail remains obscure. However, the photosystem I complex of cyanobacteria has been crystallized and found to contain three molecules of monogalactosyldiacylglycerol and one of phosphatidylglycerol. Digalactosyldiacylglycerols are required for crystallization of the light-harvesting complex II in pea chloroplasts (again together with phosphatidylglycerol). As with other biomembranes, the thylakoid membrane (where photosynthesis occurs in plants) has an asymmetric distribution of glycolipids between the two leaflets, with much of the digalactosyldiacylglycerol on the luminal leaflet. For example, there is a suggestion that the polar head group of the lipid assists the movement of protons along the luminal membrane surface to the ATPase.

It is also evident that individual glycolipids are associated in a highly specific way with various membrane proteins, where the ability of monogalactosyldiacylglycerols to form inverted micelles may be important, for example. The presence of this lipid may be required to assist the transport of proteins and other nutrients across membranes. As they are concentrated in the peribacteroid membrane surrounding nitrogen-fixing rhizobia in the nodules of legumes, they may be needed for the exchange of ammonium and nutrients, in this instance between the bacteria and the host cell. It is also evident that galactolipids are important for the lipid composition of the extra-plastid membranes in plants under phosphate-limiting conditions, assisting to conserve this important nutrient by acting as a replacement for phospholipids. The axial hydroxyl group at C4 of galactose appears to be essential for certain of these interactions and may explain why galactolipids are favoured over those containing glucose.

Mono- and digalactosylmonoacylglycerols (lyso derivatives) are found from time to time in small amounts in plant tissues. Usually the sn-1 isomer is identified, but acyl migration always occurs to give this, the more thermodynamically stable isomer. It is not clear whether these lyso-compounds play a part in galactolipid turnover and fatty acid re-modelling.

4.  Sulfoquinovosyldiacylglycerol

Sulfoquinovosyldiacylglycerol or 1,2-di-O-acyl-3-O-(6'-deoxy-6'-sulfo-α-D-glucopyranosyl)-sn-glycerol (quinovose = 6-deoxyglucose), the plant sulfolipid, is the single glycolipid most characteristic of photosynthetic organisms, including both higher plants and cyanobacteria.

 In many species of higher plants, the sn-1 position is reportedly enriched in 16:0 and the sn-2 position in 18:3 and 18:2.  Biosynthesis of the sulfoquinovose head-group involves a unique set of enzymes that serve no other function. Much remains to be learned regarding the details of the biosynthetic pathway, but it is believed that biosynthesis of the head group involves synthesis of UDP-sulfoquinovose from UDP-glucose and sulfite, followed by the transfer of sulfoquinovose to position sn-3 of 1,2-diacyl-sn-glycerols. The process occurs entirely in the plastids, although diacylglycerols transferred from the endoplasmic reticulum can be used as substrates.

Other than in active photosynthetic organisms (cyanobacteria - see below), this lipid has only been found in a few bacterial species, mainly of the genus Rhizobium, which have a symbiotic relationship with plants in root nodules and may have obtained the required genes by horizontal gene transfer. However, it was also found to comprise half the lipids of the halophilic eubacteria Planococcus sp. and Haloferax volcanii.

As with the neutral galactosyldiacylglycerols, it seems clear that the negatively charged sulfoquinovosyldiacylglycerol is important for the function of the thylakoid membrane in plants, where it is located mainly on the inner leaflet, possibly by assisting in the process of protein insertion and passage through the membranes. This membrane has a very large area, as is necessary for photosynthesis, and the sulfolipid appears to provide the required negative charge with a minimum demand for phosphate (the only phospholipid present is a small amount of phosphatidylglycerol, which appears to have a similar function). This is obviously important for plants when phosphate concentrations are limiting.

An acylated derivative of this sulfolipid, 2'-O-acyl-sulfoquinovosyldiacylglycerol has been found in the unicellular alga Chlamydomonas reinhardtii, i.e. with an additional acyl group attached to the 2'-hydroxyl of the sulfoquinovosyl head group. While the fatty acids of sulfoquinovosyldiacylglycerol were mostly saturated, the 2’-acylated analogue contained mainly unsaturated fatty acids with an 18-carbon fatty acid with four double bonds linked to the head group.

5.  Glycosyldiacylglycerols of Photosynthetic Bacteria

Cyanobacteria are oxygenic photosynthetic bacteria (Gram negative) that are distinct from most other bacteria in their lipid compositions, as they contain appreciable amounts of mono- and digalactosyldiacylglycerols together with sulfoquinovosyldiacylglycerol in which the configuration of the anomeric head groups is identical to that of the corresponding plant lipids. This may be explained by the theory that an ancestral cyanobacterial cell, which was photosynthetically active, was engulfed by a eukaryotic organism. This was the precursor of the first plant cell.

As can be seen from the data in Table 2, the overall fatty acid compositions of the lipids of the cyanobacterium Synechocystis PCC6803 resembles that of photosynthetic tissues in higher plants although the polyunsaturated fatty acids (C₁₈) are concentrated in position sn-1 in this instance with saturated (C₁₆) in position sn-2. Phosphatidylglycerol is often the only phospholipid present in appreciable amounts.

Table 2. Composition (mol %) of fatty acids in positions sn-1 and sn-2 of mono- and digalactosyl- and sulphoquinovosyldiacylglycerols from Synechocystis PCC6803*.
Position	Fatty acids
	16:0	16:1	18:0	18:1	18:2	18:3**	18:4
MGDG
sn-1	14	4	tr	-	8	54	20
sn-2	94	2	tr	2	tr	tr	tr
DGDG
sn-1	16	4	2	2	8	50	18
sn-2	94	2	2	tr	-	-	-
SQDG
sn-1	34	8	2	10	16	28	tr
sn-2	92	tr	4	tr	tr	tr	-
* Grown at 22°C; ** mainly α-18:3; tr = trace.
Data from Wada, H. and Murata, N. Plant Physiology, 92, 1062-1069 (1990).

Although the nature of the lipids is highly conserved in plants and photosynthetic bacteria, the biosynthetic mechanisms are somewhat different. In cyanobacteria, the first step in biosynthesis is the production of monoglucosyldiacylglycerol, which is then converted to the galactosyl form by an epimerization reaction. Monoglucosyldiacylglycerol is usually present at low levels only.

Many species of anoxic photosynthetic bacteria contain monogalactosyldiacylglycerols, but digalactosyldiacylglycerols are rarely found in other bacteria. However, the latter are major membrane components of free-living and bacteroid forms of Bradyrhizobium japonicum, which normally live symbiotically with plants in root nodules.

A wide variety of glycosyldiacylglycerols are found in non-photosynthetic bacteria; those with one to three glycosyl units linked to sn-1,2-diacylglycerol are most common, although others with up to five glycosyl units are found. These are very different from the plant glycosyl diacylglycerols, in that glucose is much more common than galactose, while the fatty acid components are mainly saturated, monoenoic and branched-chain or cyclopropanoid. The nature of the glucose linkages is also variable. For example, some Streptococcus species contain mono- and diglucosyldiacylglycerols, with the diglucoside unit having an α-(1→2) linkage as in kojibiose, and so can be termed ‘kojibiosyldiacylglycerols’. A galactosylglucosyldiacylglycerol is also known, as are various lipids of this type with a fatty acyl group attached to a carbohydrate moiety (usually in position 3 or 6).

Some microorganisms accumulate galactofuranosyl-diacylglycerols rather than the galactopyranosyl form, and a variety of unusual glycosyldiacylglycerols with differing carbohydrate moieties, or with differences in the glycosidic bonds from those in higher plants, have been found. For example, Micrococcus luteus synthesises mono- and dimannosyldiacylglycerols. Other bacteria have glycosyldiacylglycerols with a glycerophosphate group linked to a carbohydrate moiety (‘glycophospholipids’). Bacillus megaterium contains N-acetylgalactosamine linked to a diacylglycerol. As might be expected, even greater complexity exists in the triglycosyldiacylglycerols. In mechanistic terms, the biosynthesis of these lipids is analogous to that in higher plants described above.

In gram positive bacteria such as Staphylococcus aureus, lipoteichoic acid is anchored in the membrane by a diglucosyldiacylglycerol moiety. The membranes of this organism also contain 8 mol% of the free glycolipid, and the ratio of mono- to diglucosyldiacylglycerol may play an important role in determining bilayer stability; only the latter will form a bilayer.

Certain bacteria and algae contain the ionic 1,2-diacyl-3-O-α-D-glucuronyl-sn-glycerol among their membrane lipids, and a conjugate of this with taurine is known (see our webpage on sulfonolipids). Of course, the algal lipid illustrated has a very different fatty acid composition from those of bacteria. In addition, glucosylglucuronyl- and galacturonyldiacylglycerols have been detected in bacteria.

The complex diether isoprenoid glycerolipids from the extreme halophilic bacteria of the Archaea family exist in the form of glycosyldiacylglycerols, both as neutral lipids and in sulfated form, with two to four glycosyl units attached to glycerol.

6.  Analysis

The main neutral galactolipids in plants present no particular difficulties for analysis. They are easily separated from phospholipids by adsorption chromatography, usually by making use of the fact that they, unlike phospholipids, are soluble in acetone. Because of its highly polar acidic nature, sulfoquinovosyldiacylglycerol presents more analytical problems, but methods have been devised for its analysis that make use of adsorption or ion-exchange chromatography. Electrospray-ionization tandem mass spectrometry now appears to hold particular promise for structural analyses. The review by Heinz cited below is essential reading for anyone who wishes to study these lipids.

Recommended Reading

• Benning, C. and Ohta, H. Three enzyme systems for galactoglycerolipid biosynthesis are coordinately regulated in plants. J. Biol. Chem., 280, 2397-2400 (2005).
• Dembitsky, V.M. Astonishing diversity of natural surfactants: 3. Carotenoid glycosides and isoprenoid glycolipids. Lipids, 40, 535-557 (2005).
• Dörmann, P. and Benning, C. Galactolipids rule in seed plants. Trends Plant Sci., 7, 112-118 (2002).
• Frentzen, M. Phosphatidylglycerol and sulfoquinovosyldiacylglycerol: anionic membrane lipids and phosphate regulation. Current Opinion in Plant Biology, 7, 270-276 (2004).
• Heinz, E. Plant glycolipids: structure, isolation and analysis. In: Advances in Lipid Methodology - Three, pp. 211-332 (ed. W.W. Christie, Oily Press, Dundee) (1996).
• Hölzl, G. and Dörmann, P. Structure and function of glycoglycerolipids in plants and bacteria. Prog. Lipid Res., 46, 225-243 (2007).
• Ishizuka, I. Chemistry and functional distribution of sulfoglycolipids. Prog. Lipid Res., 36, 245-319 (1997).
• Kates, M. (ed.) Several chapters. Handbook of Lipid Research 6. Glycolipids, Phosphoglycolipids and Sulfoglycolipids, (ed. M. Kates, Plenum Press, NY) (1990).
• Pieringer, R.A. Biosynthesis of non-terpenoid lipids. In: Microbial Lipids. Volume 2, pp. 51-114 (ed. C. Ratledge and S.G. Wilkinson, Academic Press, London) (1989).
• Schmid, K.M. and Ohlrogge, J.B. Lipid metabolism in plants. In: Biochemistry of Lipids, Lipoproteins and Membranes, 4th Edition, pp. 93-126 (ed. D.E. Vance and J. Vance, Elsevier, Amsterdam) (2002).

Updated: 26/3/2008 Credits/disclaimer	Scottish Crop Research Institute (and MRS Lipid Analysis Unit), Invergowrie, Dundee (DD2 5DA), Scotland