Lipolysis of natural long chain and synthetic medium chain galactolipids by pancreatic lipase-related protein 2

Monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG) are the most abundant lipids in nature, mainly as important components of plant leaves and chloroplast membranes. Pancreatic lipase-related protein 2 (PLRP2) was previously found to express galactolipase activity, and it is assumed to be the main enzyme involved in the digestion of these common vegetable lipids in the gastrointestinal tract. Most of the previous in vitro studies were however performed with medium chain synthetic galactolipids as substrates. It was shown here that recombinant guinea pig (Cavia porcellus) as well as human PLRP2 hydrolyzed at high rates natural DGDG and MGDG extracted from spinach leaves. Their specific activities were estimated by combining the pH-stat technique, thin layer chromatography coupled to scanning densitometry and gas chromatography. The optimum assay conditions for hydrolysis of these natural long chain galactolipids were investigated and the optimum bile salt to substrate ratio was found to be different from that established with synthetic medium chains MGDG and DGDG. Nevertheless the length of acyl chains and the nature of the galactosyl polar head of the galactolipid did not have major effects on the specific activities of PLRP2, which were found to be very high on both medium chain [1786 ± 100 to 5420 ± 85 U/mg] and long chain [1756 ± 208 to 4167 ± 167 U/mg] galactolipids. Fatty acid composition analysis of natural MGDG, DGDG and their lipolysis products revealed that PLRP2 only hydrolyzed one ester bond at the sn-1 position of galactolipids. PLRP2 might be used to produce lipid and free fatty acid fractions enriched in either 16:3 n − 3 or 18:3 n − 3 fatty acids, both found at high levels in galactolipids.     

Fatty Acid Composition of Polar Lipids in Ceratodon purpureus and Pleurozium schreberi

The total amount of fatty acids in the mono- (MGDG) and diglycosyl diglyceride (DGDG) and more polar lipid fractions of frozen Ceratodon purpureus shoots was 4.6, 3.4 and 4.0 mg/g dry weight, respectively. The respective values for the tops of frozen Pleurozium schreberi were 2.6, 3.3 and 3.8 mg/g dry weight. The molar ratios MGDG/DGDG and MGDG + DGDG/chlorophyll were 1.3 and 3.7, respectively, for C. purpureus and 0.8 and 3.5 for P. schreberi. In C. purpureus the main fatty acids in the MGDG fraction were C 18:3ω3 (44% of the total fatty acids) and C 16:3ω3 (26%); in the DGDG fraction C 18:3ω3 (70%); and in the more polar lipid fraction C 18: 3ω3 (26%) and C 16:0 (25%). The proportion of C 20 polyunsaturated fatty acids was 15, 12 and 19% of the total fatty acids found in the MGDG, DGDG and more polar lipid fractions, respectively. In P. schreberi the proportion of C 20 polyunsaturated fatty acids was high in all polar lipid fractions (47, 42 and 25% in MGDG, DGDG and more polar lipid fractions, respectively). In addition, MGDG and DGDG fractions contained abundantly C 18:3ω3 (32 and 45%, respectively), and the more polar lipid fraction both C 18: 3ω3 (24%) and C 16:0 (27%).     

Chlorophyll-sensitized Photooxidation Products of Spinach Galactolipids

Spinach monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG) were oxidized with singlet molecular oxygen by the use of chlorophyll a as the photosensitizer. The oxidation products were separated from the unoxidized MGDG and DGDG by reverse-phase high performance liquid chromatography (HPLC). The products separated by HPLC were identified to be mono- and di-hydroperoxides formed by ¹O₂ oxidation of the 16:3 or 18:3 component of MGDG and DGDG. Each unsaturated fatty acid moiety in the MGDG and DGDG produced isomeric hydroperoxides in a manner similar to the corresponding fatty acid methyl ester.     

Activation of the Chloroplast Monogalactosyldiacylglycerol Synthase MGD1 by Phosphatidic Acid and Phosphatidylglycerol

One of the major characteristics of chloroplast membranes is their enrichment in galactoglycerolipids, monogalactosyldiacylglycerol (MGDG), and digalactosyldiacylglycerol (DGDG), whereas phospholipids are poorly represented, mainly as phosphatidylglycerol (PG). All these lipids are synthesized in the chloroplast envelope, but galactolipid synthesis is also partially dependent on phospholipid synthesis localized in non-plastidial membranes. MGDG synthesis was previously shown essential for chloroplast development. In this report, we analyze the regulation of MGDG synthesis by phosphatidic acid (PA), which is a general precursor in the synthesis of all glycerolipids and is also a signaling molecule in plants. We demonstrate that under physiological conditions, MGDG synthesis is not active when the MGDG synthase enzyme is supplied with its substrates only, i.e. diacylglycerol and UDP-gal. In contrast, PA activates the enzyme when supplied. This is shown in leaf homogenates, in the chloroplast envelope, as well as on the recombinant MGDG synthase, MGD1. PG can also activate the enzyme, but comparison of PA and PG effects on MGD1 activity indicates that PA and PG proceed through different mechanisms, which are further differentiated by enzymatic analysis of point-mutated recombinant MGD1s. Activation of MGD1 by PA and PG is proposed as an important mechanism coupling phospholipid and galactolipid syntheses in plants.     

Biosynthesis and desaturation of prokaryotic galactolipids in leaves and isolated chloroplasts from spinach

Mono- and digalactosyldiacylglycerol (MGDG and DGDG) were isolated from the leaves of sixteen 16:3 plants. In all of these plant species, the sn-2 position of MGDG was more enriched in C₁₆ fatty acids than sn-2 of DGDG. The molar ratios of prokaryotic MGDG to prokaryotic DGDG ranged from 4 to 10. This suggests that 16:3 plants synthesize more prokaryotic MGDG than prokaryotic DGDG. In the 16:3 plant Spinacia oleracea L. (spinach), the formation of prokaryotic galactolipids was studied both in vivo and in vitro. In intact spinach leaves as well as in chloroplasts isolated from these leaves, radioactivity from [1-¹⁴C]acetate accumulated 10 times faster in MGDG than in DGDG. After 2 hours of incorporation, most labeled galactolipids from leaves and all labeled galactolipids from isolated chloroplasts were in the prokaryotic configuration. Both in vivo and in vitro, the desaturation of labeled palmitate and oleate to trienoic fatty acids was higher in MGDG than in DGDG. In leaves, palmitate at the sn-2 position was desaturated in MGDG but not in DGDG. In isolated chloroplasts, palmitate at sn-2 similarly was desaturated only in MGDG, but palmitate and oleate at the sn-1 position were desaturated in MGDG as well as in DGDG. Apparently, palmitate desaturase reacts with sn-1 palmitate in either galactolipid, but does not react with the sn-2 fatty acid of DGDG. These results demonstrate that isolated spinach chloroplasts can synthesize and desaturate prokaryotic MGDG and DGDG. The finally accumulating molecular species, MGDG(18:3/16:3) and DGDG(18:3/16:0), are made by the chloroplasts in proportions similar to those found in leaves.     

Galactosyl headgroup interactions control the molecular packing of wheat lipids in Langmuir films and in hydrated liquid-crystalline mesophases

The behavior of the two major galactolipids of wheat endosperm, mono- (MGDG) and di-galactosyldiacylglycerol (DGDG) was studied in aqueous dispersion and at the air/liquid interface. The acyl chains of the pure galactolipids and their binary equimolar mixture are in the fluid or liquid expanded phase. SAXS measurements on liquid-crystalline mesophases associated with the electron density reconstructions show that the DGDG adopts a lamellar phase L(alpha) with parallel orientation of the headgroups with respect to the plane of the bilayer, whereas MGDG forms an inverse hexagonal phase H(II) with a specific organization of galactosyl headgroups. The equimolar mixture shows a different behavior from those previously described with formation of an Im3m cubic phase. In comparing monolayers composed of the pure galactolipids and their equimolar mixtures, PM-IRRAS spectra show significant differences in the optical properties and orientation of galactosyl groups with respect to the interface. Furthermore, Raman and FTIR spectroscopies show that the acyl chains of the galactolipid mixture are more ordered compared to those of the pure components. These results suggest strong interactions between MGDG and DGDG galactosyl headgroups and these specific physical properties of galactolipids are discussed in relation to their biological interest in wheat seed.     

Wounding stimulates the accumulation of glycerolipids containing oxophytodienoic acid and dinor-oxophytodienoic acid in Arabidopsis leaves

Although oxylipins can be synthesized from free fatty acids, recent evidence suggests that oxylipins are components of plastid-localized polar complex lipids in Arabidopsis (Arabidopsis thaliana). Using a combination of electrospray ionization (ESI) collisionally induced dissociation time-of-flight mass spectrometry (MS) to identify acyl chains, ESI triple-quadrupole (Q) MS in the precursor mode to identify the nominal masses of complex polar lipids containing each acyl chain, and ESI Q-time-of-flight MS to confirm the identifications of the complex polar lipid species, 17 species of oxylipin-containing phosphatidylglycerols, monogalactosyldiacylglycerols (MGDG), and digalactosyldiacylglycerols (DGDG) were identified. The oxylipins of these polar complex lipid species include oxophytodienoic acid (OPDA), dinor-OPDA (dnOPDA), 18-carbon ketol acids, and 16-carbon ketol acids. Using ESI triple-Q MS in the precursor mode, the accumulation of five OPDA- and/or dnOPDA-containing MGDG and two OPDA-containing DGDG species were monitored as a function of time in mechanically wounded leaves. In unwounded leaves, the levels of these oxylipin-containing complex lipid species were low, between 0.001 and 0.023 nmol/mg dry weight. However, within the first 15 min after wounding, the levels of OPDA-dnOPDA MGDG, OPDA-OPDA MGDG, and OPDA-OPDA DGDG, each containing two oxylipin chains, increased 200- to 1,000-fold. In contrast, levels of OPDA-hexadecatrienoic acid MGDG, linolenic acid (18:3)-dnOPDA MGDG, OPDA-18:3 MGDG, and OPDA-18:3 DGDG, each containing a single oxylipin chain, rose 2- to 9-fold. The rapid accumulation of high levels of galactolipid species containing OPDA-OPDA and OPDA-dnOPDA in wounded leaves is consistent with these lipids being the primary products of plastidic oxylipin biosynthesis.     

Control of galactosyl diglycerides in wheat endosperm by group 5 chromosomes

Lower levels of monogalactosyl diglyceride (MGDG) and digalactosyl diglyceride (DGDG) have been found in tetraploid wheats as compared with those in hexaploid wheats. The same difference has been found between hexaploid cultivars and tetraploid lines derived from them by D genome extraction. A lower level of MGDG and DGDG is also present in Triticum carthlicum (AABB) as compared with Aegilops squarrosa (DD) or with the synthetic T. spelta (AABBDD) obtained from them. Analysis of the appropriate nullitetrasomic and ditelosomic lines indicates that a gene or genes located in the short arm of chromosome 5D are responsible for the observed difference and that group 5 chromosomes can be ranked as to their influence on the MGDG and DGDG levels in the order 5B > 5D > 5A and 5D > 5B > 5A, respectively. These results further support our previous identification of DGDG as the lipid factor responsible for petroleum ether solubility of lipopurothionins. Since DGDG contributes to baking quality by improving the retention of fermentation gases, the present observations imply that the difference in bread-making quality between the two types of wheat is not due only to proteins contributed by the D genome.     

Expression of Fungal Cutinase and Swollenin in Tobacco Chloroplasts Reveals Novel Enzyme Functions and/or Substrates

In order to produce low-cost biomass hydrolyzing enzymes, transplastomic lines were generated that expressed cutinase or swollenin within chloroplasts. While swollenin expressing plants were homoplasmic, cutinase transplastomic lines remained heteroplasmic. Both transplastomic lines showed interesting modifications in their phenotype, chloroplast structure, and functions. Ultrastructural analysis of chloroplasts from cutinase- and swollenin-expressing plants did not show typical lens shape and granal stacks. But, their thylakoid membranes showed unique scroll like structures and chloroplast envelope displayed protrusions, stretching into the cytoplasm. Unusual honeycomb structures typically observed in etioplasts were observed in mature chloroplasts expressing swollenin. Treatment of cotton fiber with chloroplast-derived swollenin showed enlarged segments and the intertwined inner fibers were irreversibly unwound and fully opened up due to expansin activity of swollenin, causing disruption of hydrogen bonds in cellulose fibers. Cutinase transplastomic plants showed esterase and lipase activity, while swollenin transplastomic lines lacked such enzyme activities. Higher plants contain two major galactolipids, monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG), in their chloroplast thylakoid membranes that play distinct roles in their structural organization. Surprisingly, purified cutinase effectively hydrolyzed DGDG to MGDG, showing alpha galactosidase activity. Such hydrolysis resulted in unstacking of granal thylakoids in chloroplasts and other structural changes. These results demonstrate DGDG as novel substrate and function for cutinase. Both MGDG and DGDG were reduced up to 47.7% and 39.7% in cutinase and 68.5% and 67.5% in swollenin expressing plants. Novel properties and functions of both enzymes reported here for the first time should lead to better understanding and enhanced biomass hydrolysis.     

Biosynthesis of sulfoquinovosyldiacylglycerol in higher plants: the origin of the diacylglycerol moiety

The positional distribution of fatty acids in chloroplast polar lipids and phosphatidylcholine from leaves of four plants has been measured in order to determine the origin of the diacylglycerol (DAG) moieties of each lipid. In spinach and tobacco, the DAG of sulfoquinovosyldiacylglycerol (SQDG), monogalactosyldiacylglycerol (MGDG) and digalactosylglycerol (DGDG) were derived partly from the chloroplast and partly from the cytoplasm. The contribution of the chloroplast pathway differed for each lipid, but in both plants the proportion of a lipid derived from that pathway was in the order SQDG greater than MGDG greater than DGDG. In contrast, all the DAG moieties of the three glycolipids of wheat and cucumber were derived from the cytoplasm. The DAG moiety of chloroplast phosphatidylglycerol was synthesized in the chloroplast in all four plants.     

Galactosyl headgroup interactions control the molecular packing of wheat lipids in Langmuir films and in hydrated liquid-crystalline mesophases

The behavior of the two major galactolipids of wheat endosperm, mono- (MGDG) and di-galactosyldiacylglycerol (DGDG) was studied in aqueous dispersion and at the air/liquid interface. The acyl chains of the pure galactolipids and their binary equimolar mixture are in the fluid or liquid expanded phase. SAXS measurements on liquid-crystalline mesophases associated with the electron density reconstructions show that the DGDG adopts a lamellar phase L_α with parallel orientation of the headgroups with respect to the plane of the bilayer, whereas MGDG forms an inverse hexagonal phase H_II with a specific organization of galactosyl headgroups. The equimolar mixture shows a different behavior from those previously described with formation of an Im3m cubic phase. In comparing monolayers composed of the pure galactolipids and their equimolar mixtures, PM-IRRAS spectra show significant differences in the optical properties and orientation of galactosyl groups with respect to the interface. Furthermore, Raman and FTIR spectroscopies show that the acyl chains of the galactolipid mixture are more ordered compared to those of the pure components. These results suggest strong interactions between MGDG and DGDG galactosyl headgroups and these specific physical properties of galactolipids are discussed in relation to their biological interest in wheat seed.     

Functional roles of the major chloroplast lipids in the violaxanthin cycle

Monogalactosyldiacylglyceride (MGDG) and digalactosyldiacylglyceride (DGDG) are the major membrane lipids of chloroplasts. The question of the specialized functions of these unique lipids has received limited attention. One function is to support violaxanthin de-epoxidase (VDE) activity, an enzyme of the violaxanthin cycle. To understand better the properties of this system, the effects of galactolipids and phosphatidylcholines on VDE activity were examined by two independent methods. The results show that the micelle-forming lipid (MGDG) and bilayer forming lipids (DGDG and phosphatidylcholines) support VDE activity differently. MGDG supported rapid and complete de-epoxidation starting at a threshold lipid concentration (10 μM) coincident with complete solubilization of violaxanthin. In contrast, DGDG supported slow but nevertheless complete to nearly complete de-epoxidation at a lower lipid concentration (6.7 μM) that did not completely solubilize violaxanthin. Phosphotidylcholines showed similar effects as DGDG except that de-epoxidation was incomplete. Since VDE requires solubilized violaxanthin, aggregated violaxanthin in DGDG at low concentration must become solubilized as de-epoxidation proceeds. High lipid concentrations had lower activity possibly due to formation of multilayered structures (liposomes) that restrict accessibility of violaxanthin to VDE. MGDG micelles do not present such restrictions. The results indicate VDE operates throughout the lipid phase of the single bilayer thylakoid membrane and is not limited to putative MGDG micelle domains. Additionally, the results also explain the differential partitioning of violaxanthin between the envelope and thylakoid as due to the relative solubilities of violaxanthin and zeaxanthin in MGDG, DGDG and phospholipids. The violaxanthin cycle is hypothesized to be a linked system of the thylakoid and envelope for signal transduction of light stress.     

Mineral supplementation of white wheat flour is necessary to maintain adequate mineral status and bone characteristics in rats

This experiment was designed to compare the effect of ingestion of a wheat flours on mineral status and bone characteristics in rats. White flour was tested either without further mineral supplementation or with Mg, Fe, Zn and Cu supplementation. The flour diets were compared to a control purified diet. Four groups of 10 male Wistar rats each were fed one of the experimental diets for 6 wk and mineral status and tissue retention as well as bone characteristics were determined. As expected, mineral intake, except for calcium, was significantly lesser in rats fed the white flour diet than in the other groups. The rats fed the white flour diet had the lowest food intake, weight gain, fecal excretion and intestinal fermentation. The most important result was that Mg and Fe status were drastically lower in rats fed the white flour diet than in those fed whole flour or control diets. The status of these both elements were significantly improved by the mineral supplementation of white flour. There were no major significant differences between mineral-supplemented white flour and whole flour groups in mineral status. Furthermore, bone mineral densities (total, metaphyseal and diphyseal) were significantly lower in rats fed white flour diet compared to the other diet groups, while no significant difference was observed between the mineral-supplemented white flour, whole flour or control diet groups. In conclusion, the present work shows clearly the importance of mineral-supplementation of white wheat flour to sustain an adequate intake of minerals. Our results indicate also that the whole wheat flour did not negatively alter mineral bioavailability, in comparison to mineral supplemented white flour. Clinical studies are still needed to confirm these rat results in human.     

Glycolipid and phospholipid composition of wheat and barley chloroplasts

Composition of complex lipid of chloroplasts of two cultivars of wheat and barley was determined at tillering, ear emergence and grain filling stages. The chloroplast lipids, MGDG, DGDG and PG were maximum at grain filling stage in both wheat and barley. PC content showed variations at different stages in both the crops while no significant changes were observed in PI concentrations at grain filling stage.     

Role of hexagonal structure-forming lipids in diadinoxanthin and violaxanthin solubilization and de-epoxidation

In this study, we have examined the influence of different lipids on the solubility of the xanthophyll cycle pigments diadinoxanthin (Ddx) and violaxanthin (Vx) and on the efficiency of Ddx and Vx de-epoxidation by the enzymes Vx de-epoxidase (VDE) from wheat and Ddx de-epoxidase (DDE) from the diatom Cyclotella meneghiniana, respectively. Our results show that the lipids MGDG and PE are able to solubilize both xanthophyll cycle pigments in an aqueous medium. Substrate solubilization is essential for de-epoxidase activity, because in the absence of MGDG or PE Ddx and Vx are present in an aggregated form, with limited accessibility for DDE and VDE. Our results also show that the hexagonal structure-forming lipids MGDG and PE are able to solubilize Ddx and Vx at much lower lipid concentrations than bilayer-forming lipids DGDG and PC. We furthermore found that, in the presence of MGDG or PE, Ddx is much more solubilizable than Vx. This substantial difference in Ddx and Vx solubility directly affects the respective de-epoxidation reactions. Ddx de-epoxidation by the diatom DDE is saturated at much lower MGDG or PE concentrations than Vx de-epoxidation by the higher-plant VDE. Another important result of our study is that bilayer-forming lipids DGDG and PC are not able to induce efficient xanthophyll de-epoxidation. Even in the presence of high concentrations of DGDG or PC, where Ddx and Vx are completely solubilized, a strongly inhibited Ddx de-epoxidation is observed, while Vx de-epoxidation by VDE is completely absent. This indicates that the inverted hexagonal phase domains provided by lipid MGDG or PE are essential for de-epoxidase activity. We conclude that in the natural thylakoid membrane MGDG serves to solubilize the xanthophyll cycle pigments and furthermore provides inverted hexagonal structures associated with the membrane bilayer, which are essential for efficient xanthophyll de-epoxidase activity.     

Glycolipids of turions and leaves of Utricularia vulgaris at different stages of development

Turions of Utricularia vulgaris L. were germinated in long-day conditions at 15°C for 1,3 and 6 days and their glycolipid composition was compared with that of resting but vernalized turions. Digalactosyldiacylglycerides (DGDG), monogalactosyldiacylglycerides (MGDG) and cerebrosides were present at all stages of development. No great changes were found in the glycolipid classes during sprouting but some differences were noted in the proportions of fatty acids. The most common fatty acids in all three glycolipid classes studied were 16:0, 18:0 and 18:2. MGDG and DGDG also contained relatively much 18:3 and its proportion increased during germination. Young turions and full-grown leaves collected from nature contained the same glycolipid classes as the sprouting turions. The developmental stage of the organs studied is reflected in the fatty acid composition of DGDG and MGDG but is not so evident in the cerebrosides. The 18:2 fatty acid is rather typical of the resting turions, especially in DGDG.     

The effect of light intensity on galactolipid synthesis in Vicia faba leaves

The effect of light intensity upon galactolipid synthesis in Vicia faba leaf tissue was studied at two CO₂ concentrations, 0.03 and 1%. The rates of galactolipid synthesis were estimated by determining the amount of radioactivity in each of the two galactoses of digalactosyl diacylglycerol (DGDG) and the single galactose of monogalactosyl diacylglycerol (MGDG), a technique based upon the accepted pathway for galactolipid synthesis in which galactosylation is the terminal step in biosynthesis. The results suggest that the rates of MGDG and DGDG synthesis were similar under all conditions and that galactolipid synthesis was not directly affected by light intensity. The quantity of radioactivity incorporated into the galactoses of individual molecular species of MGDG and DGDG were similar under the light conditions used.     

Mono‐ and digalactosyldiacylglycerol composition of glaucocystophytes (Glaucophyta): A modern interpretation using positive‐ion electrospray ionization/mass spectrometry/mass spectrometry

Glaucocystophytes are freshwater algae that possess an almost‐intact cyanobacterium, referred to as a cyanelle, as their photosynthetic organelle. Because the cyanelle represents an intermediate state in plastid evolution, glaucocystophytes have been the subject of several studies to characterize the genetics and biochemistry of their cyanelles. However, only a small handful of older studies exist on the composition of their lipids, particularly two major plastid lipids, mono‐ and digalactosyldiacylglycerol (MGDG and DGDG, respectively), found in all photosynthetic life. Our study has used a modern mass spectrometry approach, namely positive‐ion electrospray ionization/mass spectrometry/mass spectrometry, to provide a fresh interpretation of the MGDG and DGDG composition of the species, Cyanophora paradoxa Korshikov and Glaucocystis nostochinearum Itzigsohn, representing two glaucocystophyte genera. We have found that the major forms of MGDG and DGDG (with sn‐1/sn‐2 regiochemistry) are 20:5/16:0 MGDG, 20:5/20:5 MGDG, 20:5/16:0 DGDG, and 20:5/20:5 DGDG. A comparison of these four forms, along with other more minor forms of MGDG and DGDG, to two examples of cyanobacteria has revealed that glaucocystophytes do not share intact forms of MGDG and DGDG with extant cyanobacteria, but may have maintained certain C₁₆ and C₁₈ cyanobacterial fatty acids.     

Synthesis of digalactosyl diacylglycerols and their structure-inhibitory activity on human lanosterol synthase

Digalactosyl and monogalactocyl diacylglycerols (DGDG and MGDG), which were identified as anti-hyperlipemia active components in Colocasia esculenta (Taro), were synthesized. The inhibitory activity of DGDG, MGDG and related compounds on human lanosterol synthase was evaluated as anti-hyperlipemic activity. DGDG with two myristoyl groups at both sn-1 and sn-2 positions and with an oleoyl group at the sn-1 position showed the most potent activity.     

Synthesis of mono- and digalactosyldiacylglycerol in isolated spinach chloroplasts

Purified, intact chloroplasts of Spinacia oleracea L. synthesize galactose-labeled mono- and digalactosyldiacylglycerol (MGDG and DGDG) from UDP-[U-¹⁴C]galactose. In the presence of high concentrations of unchelated divalent cations they also synthesize tri- and tetra-galactosyldiacylglycerol. The acyl chains of galactose-labeled MGDG are strongly desaturated and such MGDG is a good precursor for DGDG and higher oligogalactolipids. The synthesis of MGDG is catalyzed by UDP-Gal:sn-1,2-diacylglycerol galactosyltransferase, and synthesis of DGDG and the oligogalactolipids is exclusively catalyzed by galactolipid:galactolipid galactosyltransferase. The content of diacylglycerol in chloroplasts remains low during UDP-Gal incorporation. This indicates that formation of diacylglycerol by galactolipid:galactolipid galactosyltransferase is balanced with diacylglycerol consumption by UDP-Gal:diacylglycerol galactosyltransferase for MGDG synthesis. Incubation of intact spinach chloroplasts with [2-¹⁴C]acetate or sn-[U-¹⁴C]glycerol-3-P in the presence of Mg²⁺ and unlabeled UDP-Gal resulted in high ¹⁴C incorporation into MGDG, while DGDG labeling was low. This de novo made MGDG is mainly oligoene. Its conversion into DGDG is also catalyzed, at least in part, by galactolipid:galactolipid galactosyltransferase.