Galactolipid Galactosyltransferase Research Articles

SENSITIVE TO FREEZING2 is crucial for growth of Marchantia polymorpha under acidic conditions.

Land plants have evolved many systems to adapt to a wide range of environmental stresses. In seed plants, oligogalactolipid synthesis is involved in tolerance to freezing and dehydration, but it has not been analyzed in non-vascular plants. Here we analyzed trigalactosyldiacylglycerol (TGDG) synthesis in Marchantia polymorpha. TGDG is synthesized by galactolipid: galactolipid galactosyltransferase [GGGT; SENSITIVE TO FREEZING2 (SFR2) in Arabidopsis]. We analyzed the subcellular localization and GGGT activity of two M. polymorpha SFR2 homologs (MpGGGT1 and MpGGGT2, each as a GFP-fusion protein) using a transient expression system in Nicotiana benthamiana leaves and found that MpGGGT1-GFP localized in the chloroplast envelope membrane. We produced mutants Mpgggt1 and Mpgggt2 and found that TGDG did not accumulate in Mpgggt1 upon treatment of the thallus with acetic acid. Moreover, growth of Mpgggt1 mutants was impaired by acetic acid treatment. Microscopy revealed that the acetic acid treatment of M. polymorpha plants damaged intracellular membranes. The fact that the effect was similar for wild-type and Mpgggt1 plants suggested that MpGGGT has a role in recovery from damage. These results indicate that MpGGGT plays a crucial role in M. polymorpha growth under conditions of acid stress, which may have been encountered during the ancient terrestrial colonization of plants.

A family 1 galactosyl hydrolase performs galactosyltransferase activity requiring Mg2+ and conferring freezing tolerance (617.8)

The two most abundant photosynthetic thylakoid membrane lipids are monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG). Understanding galactolipid metabolism is therefore critical to understanding thylakoid formation and subsequent photosynthetic activity. The first enzymatic activity described to synthesize DGDG was named galactolipid: galactolipid galactosyl transferase (GGGT), as it removed a galactosyl head group from MGDG and transferred it to another MGDG making DGDG. Though it is now understood that a separate enzyme, DGD1, synthesizes most of the DGDG in vivo, GGGT was re‐identified in Arabidopsis as the gene product of SENSITIVE TO FREEZING 2 (SFR2). SFR2 was shown to protect Arabidopsis from freezing damage, to be located on the chloroplast outer envelope and to be similar to family‐1 β‐glycosidases. Here, we present a model of the 3‐dimensional structure of SFR2 based on its homology to family‐1 β‐glycosidases and show the functional significance of several unique regions. Using the model, we identify residues required for SFR2 activity that are not conserved among family‐1 β‐glycosidases. We experimentally define pH and temperature optimums for enzymatic activity in vitro. Unexpectedly, we show that SFR2 is a metalloenzyme, unlike its family members. Our data suggest that while SFR2 retains the core structure and active site residues of a family‐1 β‐glycosidase, it has unique residues, regions, and co‐factors required to alter its function to that of a transferase.Grant Funding Source: Supported by the Department of Energy

Rice Os9BGlu31 Is a Transglucosidase with the Capacity to Equilibrate Phenylpropanoid, Flavonoid, and Phytohormone Glycoconjugates

Glycosylation is an important mechanism of controlling the reactivities and bioactivities of plant secondary metabolites and phytohormones. Rice (Oryza sativa) Os9BGlu31 is a glycoside hydrolase family GH1 transglycosidase that acts to transfer glucose between phenolic acids, phytohormones, and flavonoids. The highest activity was observed with the donors feruloyl-glucose, 4-coumaroyl-glucose, and sinapoyl-glucose, which are known to serve as donors in acyl and glucosyl transfer reactions in the vacuole, where Os9BGlu31 is localized. The free acids of these compounds also served as the best acceptors, suggesting that Os9BGlu31 may equilibrate the levels of phenolic acids and carboxylated phytohormones and their glucoconjugates. The Os9BGlu31 gene is most highly expressed in senescing flag leaf and developing seed and is induced in rice seedlings in response to drought stress and treatment with phytohormones, including abscisic acid, ethephon, methyljasmonate, 2,4-dichlorophenoxyacetic acid, and kinetin. Although site-directed mutagenesis of Os9BGlu31 indicated a function for the putative catalytic acid/base (Glu(169)), catalytic nucleophile residues (Glu(387)), and His(386), the wild type enzyme displays an unusual lack of inhibition by mechanism-based inhibitors of GH1 β-glucosidases that utilize a double displacement retaining mechanism.

Disruption of the two digalactosyldiacylglycerol synthase genes DGD1 and DGD2 in Arabidopsis reveals the existence of an additional enzyme of galactolipid synthesis.

Two genes (DGD1 and DGD2) are involved in the synthesis of the chloroplast lipid digalactosyldiacylglycerol (DGDG). The role of DGD2 for galactolipid synthesis was studied by isolating Arabidopsis T-DNA insertional mutant alleles (dgd2-1 and dgd2-2) and generating the double mutant line dgd1 dgd2. Whereas the growth and lipid composition of dgd2 were not affected, only trace amounts of DGDG were found in dgd1 dgd2. The growth and photosynthesis of dgd1 dgd2 were affected more severely compared with those of dgd1, indicating that the residual amount of DGDG in dgd1 is crucial for normal plant development. DGDG synthesis was increased after phosphate deprivation in the wild type, dgd1, and dgd2 but not in dgd1 dgd2. Therefore, DGD1 and DGD2 are involved in DGDG synthesis during phosphate deprivation. DGD2 was localized to the outer side of chloroplast envelope membranes. Like DGD2, heterologously expressed DGD1 uses UDP-galactose for galactosylation. Galactolipid synthesis activity for monogalactosyldiacylglycerol (MGDG), DGDG, and the unusual oligogalactolipids tri- and tetragalactosyldiacylglycerol was detected in isolated chloroplasts of all mutant lines, including dgd1 dgd2. Because dgd1 and dgd2 carry null mutations, an additional, processive galactolipid synthesis activity independent from DGD1 and DGD2 exists in Arabidopsis. This third activity, which is related to the Arabidopsis galactolipid:galactolipid galactosyltransferase, is localized to chloroplast envelope membranes and is capable of synthesizing DGDG from MGDG in the absence of UDP-galactose in vitro, but it does not contribute to net galactolipid synthesis in planta.

DGD2, an Arabidopsis Gene Encoding a UDP-Galactose-dependent Digalactosyldiacylglycerol Synthase Is Expressed during Growth under Phosphate-limiting Conditions

The galactolipid digalactosyldiacylglycerol (DGDG), one of the main chloroplast lipids in higher plants, is believed to be synthesized by the galactolipid:galactolipid galactosyltransferase, which transfers a galactose moiety from one molecule of monogalactosyldiacylglycerol (MGDG) to another. Here, we report that Arabidopsis as well as other plant species contain two genes, DGD1 and DGD2, encoding enzymes with DGDG synthase activity. Using MGDG and UDP-galactose as substrates for in vitro assays with DGD2 we could for the first time measure DGDG synthase activity of a heterologously expressed plant cDNA. UDP-galactose, but not MGDG, serves as the galactose donor for DGDG synthesis catalyzed by DGD2, providing clear evidence for the existence of a UDP-galactose-dependent DGDG synthase in higher plants. In in vitro assays, DGD2 was capable of galactosylating DGDG, resulting in the synthesis of an oligogalactolipid tentatively identified as trigalactosyldiacylglycerol. DGD2 mRNA expression in leaves was very low but was strongly induced during growth under phosphate-limiting conditions. This induction correlates with the previously described increase in DGDG during phosphate deprivation. Therefore, in contrast to DGD1, which is responsible for the synthesis of the bulk of DGDG found in chloroplasts, DGD2 apparently is involved in the synthesis of DGDG under specific growth conditions.

Lipid synthesis and metabolism in the plastid envelope

Plastid envelope membranes play a major role in the biosynthesis of glycerolipids. In addition, plastids are characterized by the occurrence of plastid‐specific membrane glycolipids (galactolipids, a sulfolipid). Plant lipid metabolism therefore has unique features, when compared to that of other eukaryotic organisms, such as animals and yeast. However, the glycerolipid biosynthetic pathway in chloroplasts is almost identical to that found in cyanobacteria, and reflects the prokaryotic origin of the chloroplast. Fatty acids generated in the plastid stroma are substrates for a whole set of enzymes involved in the synthesis of polar lipids of plastid membranes such as galactolipids, the sulfolipid, the phosphatidylglycerol. In addition, fatty acids are exported outside the plastid where they are used for extraplastidial polar lipid synthesis (phosphatidylcholine, phosphatidylethanolamine, etc.). Various desaturation steps leading to the formation of polyunsaturated fatty acids occur in various cell compartments, especially in chloroplasts, using fatty acids esterified to polar lipids as substrates. Furthermore, plant glycerolipids can be metabolized by a series of very active envelope enzymes, such as the galactolipid:galactolipid galactosyltransferase and the acyl‐galactolipid forming enzyme. The physiological significance of these enzymes is however largely unknown. One of the most active pathways involved in lipid metabolism and present in envelope membranes is the oxylipin pathway: polyunsaturated fatty acids that are released from polar lipids under various conditions (injury, pathogen attack) are converted to oxylipin. Thus, the plastid envelope membranes are also involved in the formation of signalling molecules.

Feedback inhibition of phosphatidate phosphatase from spinach chloroplast envelope membranes by diacylglycerol.

Because the envelope phosphatidate phosphatase plays a pivotal role in chloroplast glycerolipid metabolism, we have analyzed whether diacylglycerol could be a regulatory factor of the enzyme. Using isolated envelope membranes in which the level of diacylglycerol was modified by thermolysin treatment of intact chloroplasts to destroy the galactolipid:galactolipid galactosyltransferase, we have demonstrated that phosphatidate phosphatase activity was reduced when the membrane was enriched in diacylglycerol. All 1,2-diacylglycerol molecular species assayed were demonstrated to inhibit the enzyme to about the same extent. Kinetic studies with envelope from thermolysin-treated chloroplasts were performed in the absence and presence of diacylglycerol, and diacylglycerol was shown to be a powerful competitive inhibitor of the reaction. Finally, using isolated intact spinach chloroplasts, we have demonstrated that in situ phosphatidate phosphatase activity can be modulated by the level of diacylglycerol present in the membrane. The relevance of phosphatidate phosphatase inhibition by diacylglycerol in the regulation of chloroplast glycerolipid biosynthesis is discussed.

Activities of glutathione reductase and superoxide dismutase in relation to changes of lipids and pigments due to ozone in seedlings of Pinus sylvestris (L.)

Scots pine seedlings (Pinus sylvestris L.) were exposed to ozone at a high-level, 300 ppb 8 h/day for 5 days, and at a low-level, 70-80 ppb 8 h/day for 10 days. In the high-level and low-level experiment the chlorophyll a content decreased 11% and 8%, respectively. The high-level ozone experiment affected the xanthophyll cycle by a 19% higher level of violaxanthin in the exposed seedlings compared to controls at day 5. This was also accompanied by a 42% lower concentration of zeaxanthin. No significant changes of the carotenoids neoxanthin, lutein, alpha-carotene or beta-carotene were found. The molar ratio monogalactosyl diacylglycerol/digalactosyl diacylglycerol decreased by 11% in the high-level ozone exposure. This suggests that the galactolipid:galactolipid galactosyl transferase, located in the outer envelope membrane of the chloroplast, was stimulated by ozone. No significant changes in the total membrane lipid or the acyl group composition was detected. In the high-level ozone experiment the concentration of soluble protein increased in the exposed seedlings during the last 3 days of the experimental period without any change in total protein. No visible damages were observed. The results showed that the activity of the defence enzymes superoxide dismutase (EC 1.15.1.1) and glutathione reductase (EC 1.6.4.2) was not altered after ozone exposure.

Pathway for the Synthesis of Triacylglycerols from Monogalactosyldiacylglycerols in Ozone-Fumigated Spinach Leaves

When the upper leaf surface of spinach (Spinacia oleracea L.) plants was treated with [1-(14)C]acetate and grown for 2 days, (14)C was effectively incorporated into acyl moieties of leaf lipids in ratios approximately their composition by mass. Fumigation of the plants with ozone (0.5 microliter per liter) caused a redistribution of (14)C among lipid classes, i.e. a marked increase of (14)C content in triacylglycerol (TG) and 1,2-diacylglycerol (1,2-DG) and a decrease of label in monogalactosyldiacylglycerol (MGDG) without affecting (14)C distribution in leaf fatty acids. Label in both TG and 1,2-DG was found predominantly in their polyene molecular species. Since MGDG consists of similar polyene molecular species, the results indicate the synthesis of TG from MGDG via 1,2-DG. Label was also accumulated in tri- and tetragalactosyldiacylglycerol, products of galactolipid:galactolipid galactosyltransferase (GGGT). Moreover, there was a close relation between increases in the amounts of TG and the oligogalactolipids in ozonetreated leaves. These results indicate that MGDG was converted to 1,2-DG by GGGT and then to TG. In intact chloroplasts isolated from ozone-treated leaves, there was an enhanced production of free fatty acid (FFA), which was diminished by the addition of coenzyme A (CoA) and ATP, indicating that ozone stimulated the hydrolysis of MGDG to liberate FFA, which was in turn converted to acyl-CoA. The final step of TG synthesis, acylation of 1,2-DG with acyl-CoA, was confirmed by feeding with [1-(14)C]linolenic acid in leaf discs excised from ozone-fumigated leaves; (14)C was effectively incorporated into TG but not into 1,2-DG. These results demonstrate the synthesis of TG from 1,2-DG and FFA which were liberated from MGDG in ozone-fumigated spinach leaves.

Free Fatty Acids Regulate Two Galactosyltransferases in Chloroplast Envelope Membranes Isolated from Spinach Leaves

Effects of MgCl(2) and free fatty acids (FFA) on galactolipid:galactolipid galactosyltransferase (GGGT) and UDP-galactose: 1,2-diacylglycerol galactosyltransferase (UDGT) in chloroplast envelope membranes isolated from spinach (Spinacia oleracea L.) leaves were examined. GGGT activity was sigmoidally stimulated by MgCl(2) with a saturated concentration of more than 5 millimolar. Free alpha-linolenic acid (18:3) caused a drastic increase in GGGT activity under limiting concentrations of MgCl(2), without affecting its maximum activity at higher MgCl(2) concentrations. Free 18:3 alone did not affect the GGGT activity. The effective species of FFA for the stimulation of GGGT activity in the presence of MgCl(2) were unsaturated 16- and 18-carbon fatty acids. GGGT activity was also stimulated by 18:3 in the presence of MnCl(2), CaCl(2) and a high concentration of KCl in place of MgCl(2). UDGT activity was hyperbolically enhanced by MgCl(2) with a saturated concentration of 1 to 2 millimolar. In contrast to GGGT, UDGT was severely inhibited by 18:3, and MgCl(2)-induced stimulation was completely abolished by 18:3. Unsaturated 16- and 18-carbon fatty acids were more inhibitory to UDGT than the saturated acids. The dependence of GGGT activity on monogalactosyldiacylglycerol (MGDG) and MgCl(2) concentrations was identical in the envelope membranes isolated from non- and ozone (0.5 microliter/liter)-fumigated spinach leaves, indicating that GGGT remained active in the leaves during ozone fumigation. The results are discussed in relation to the regulation of galactolipid biosynthesis by the endogenous FFA in the envelopes and to the involvement of GGGT in the triacylglycerol synthesis from MGDG in ozone-fumigated leaves.

Biosynthesis of Digalactosyldiacylglycerol in Plastids from 16:3 and 18:3 Plants

Intact chloroplasts isolated from leaves of eight species of 16:3 and 18:3 plants and chromoplasts isolated from Narcissus pseudonarcissus L. flowers synthesize galactose-labeled mono-, di-, and trigalactosyldiacylglycerol (MGDG, DGDG, and TGDG) when incubated with UDP-[6-(3)H]galactose. In all plastids, galactolipid synthesis, and especially synthesis of DGDG and TGDG, is reduced by treatment of the organelles with the nonpenetrating protease thermolysin. Envelope membranes isolated from thermolysin-treated chloroplasts of Spinacia oleracea L. (16:3 plant) and Pisum sativum L. (18:3 plant) or membranes isolated from thermolysin-treated chromoplasts are strongly reduced in galactolipid:galactolipid galactosyltransferase activity, but not with regard to UDP-Gal:diacylglycerol galactosyltransferase. For the intact plastids, this indicates that thermolysin treatment specifically blocks DGDG (and TGDG) synthesis, whereas MGDG synthesis is not affected. Neither in chloroplast nor in chromoplast membranes is DGDG synthesis stimulated by UDP-Gal. DGDG synthesis in S. oleracea chloroplasts is not stimulated by nucleoside 5'-diphospho digalactosides. Therefore, galactolipid:galactolipid galactosyltransferase is so far the only detectable enzyme synthesizing DGDG. These results conclusively suggest that the latter enzyme is located in the outer envelope membrane of different types of plastids and has a general function in DGDG synthesis, both in 16:3 and 18:3 plants.

Preparation and Characterization of Envelope Membranes from Nongreen Plastids

We have developed a reliable procedure for the purification of envelope membranes from cauliflower (Brassica oleracea L.) bud plastids and sycamore (Acer pseudoplatanus L.) cell amyloplasts. After disruption of purified intact plastids, separation of envelope membranes was achieved by centrifugation on a linear sucrose gradient. A membrane fraction, having a density of 1.122 grams per cubic centimeter and containing carotenoids, was identified as the plastid envelope by the presence of monogalactosyldiacylglycerol synthase. Using antibodies raised against spinach chloroplast envelope polypeptides E24 and E30, we have demonstrated that both the outer and the inner envelope membranes were present in this envelope fraction. The major polypeptide in the envelope fractions from sycamore and cauliflower plastids was identified immunologically as the phosphate translocator. In the envelope membranes from cauliflower and sycamore plastids, the major glycerolipids were monogalactosyldiacylglycerol, digalactosyldiacylglycerol, and phosphatidylcholine. Purified envelope membranes from cauliflower bud plastids and sycamore amyloplasts also contained a galactolipid:galactolipid galactosyltransferase, enzymes for phosphatidic acid and diacylglycerol biosynthesis, acyl-coenzyme A thioesterase, and acyl-coenzyme A synthetase. These results demonstrate that envelope membranes from nongreen plastids present a high level of homology with chloroplasts envelope membranes.

UDPgalactose-independent synthesis of monogalactosyldiacylglycerol. An enzymatic activity of the spinach chloroplast envelope

Envelope membranes isolated from spinach chloroplasts synthesize monogalactosyldiacylglycerol (MGDG) from sn-1,2-diacylglycerol in the absence of UDPgalactose. The enzyme responsible galactosylates di[1- 14C]oleoylglycerol and diacyl[2- 3H]glycerol with rates comparable to the rate of UDPgalactose:di-acylglycerol galactosyltransferase (EC 2.4.1.46), if the latter enzyme is measured separately from other galactosyltransferase activities (Heemskerk J.W.M. et al. (1987) Biochim. Biophys. Acta 918, 189–203). The velocity of UDPgalactose-independent synthesis of MGDG increases considerably with the concentration of diacylglycerol, the maximal velocity being 550 nmol/mg envelope protein per h. A galactolipid, probably digalactosyldiacylglycerol (DGDG), serves as galactosyl donor. The reaction is not affected by the presence of UDP, Mg 2+ or EDTA. It is inhibited by treatment of the chloroplasts with the proteinase thermolysin, indicating a localization in the outer envelope membrane. Together, these results suggest that UDPgalactose-independent synthesis of MDGD is catalyzed by reversible action of galactolipid:galactolipid galactosyltransferase (EC 2.4.1.-). The enzymatic reaction may provide a separate pathway for the synthesis of MDGD in vivo, but can also be understood as a mechanism to maintain a low level of diacylglycerol in the envelope membrane or, alternatively, as a way to facilitate transfer of galactosyl groups between galactolipid molecules.

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-(14)C]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-(14)C]acetate or sn-[U-(14)C]glycerol-3-P in the presence of Mg(2+) and unlabeled UDP-Gal resulted in high (14)C 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.

Characterization of galactosyltransferases in spinach chloroplast envelopes

Two galactosyltransferases involved in the galactolipid metabolism of spinach chloroplast envelopes were studied by specific assays: UDPgalactose: 1,2-diacylglycerol galactosyltransferase, which synthetizes monogalactosyldiacylglycerol from UDPgalactose plus diacylglycerol; and galactolipid: galactolipid galactosyltransferase (GGGT), which forms di- , tri- and tetragalactosyldiacylglycerol by dismutation of galactosyl groups between two galactosyldiacylglycerols. In the assay developed for UDPgalactose: 1,2-diacylglycerol galactosyltransferase, chloroplast envelope membranes were mixed with liposomes of phosphatidylcholine, and the mixture was treated with phospholipase C (from Bacillus cereus). The diacylglycerol produced from phosphatidylcholine is used by UDPgalactose: 1,2-diacylglycerol galactosyltransferase for synthesis of monogalactosyldiacylglycerol in the presence of UDPgalactose. Several characteristics of this enzyme were studied, including the effect of substrate concentrations, pH and temperature. UDPgalactose: 1,2-di-acylglycerol galactosyltransferase did not require cations for activity, but was stimulated by Mg 2+ and Mn 2+. Of the mono- and divalent cations tested only Zn 2+, Cd 2+ and Fe 2+ were inhibitory. Specific inhibitors for UDPgalactose: 1,2-diacylglycerol galactosyltransferase were UDP and N-ethylmaleimide. In contrast to UDPgalactose: 1,2-diacylglycerol galactosyltransferase, galactolipid: galactolipid galactosyltransferase was strongly stimulated by a series of mono- and divalent cations, most stimulatory being Mn 2+ Ba 2+, Ca 2+ and Mg 2+. Galactolipid: galactolipid galactosyltransferase was specifically inhibited by low concentrations of Zn 2+ (1 mM) and by chelating anions. UDPgalactose: 1,2-diacylglycerol galactosyltransferase synthetized monogalactosyldiacylglycerol from various molecular species of diacylglycerol; the highest activity was measured with distearoylglycerol and dioleoylglycerol. On the other hand, digalactosyldiacylglycerol synthesis by galactolipid: galactolipid galactosyltransferase proceeded most rapidly by galactosyl transfer to hexaene species of monogalactosyldiacylglycerol.

Localization of galactolipid: galactolipid galactosyltransferase and acyltransferase in outer envelope membrane of spinach chloroplasts

We have measured the localization within spinach chloroplast envelope membranes of two galactolipidmanipulating enzymes, the galactolipid:galactolipid galactosyltransferase and the galactolipid:galactolipid acyltransferase. Both are localized on the outer envelope membrane. This situation differs strikingly from the localization of the enzymes involved in monogalactosyldiacylglycerol synthesis, on the inner envelope membrane.

Separation of chloroplast polar lipids and measurement of galactolipid metabolism by high-performance liquid chromatography

Procedures are described for the separation of polar lipids from plant chloroplasts by high-performance liquid chromatography, using a polar-modified silica column. Glycolipids and phospholipids were eluted with a gradient of 2-propanol/ n-hexane (80:55, v v ) and 2-propanol/ n-hexane/water/methanol (80:55:15:10, v v ). The lipids were detected by uv absorbance at 202 nm. Diacylglycerol and mono-, di-, and trigalactosyldiacylglycerol and phosphatidylcholine were separated on a LiChrosorb NH 2 column (7-μm particles, Merck, FRG), but acidic lipids were retained. These lipids could be quantified from their 202-nm absorbance recording. The absorption coefficients obtained depended on the mean number of double bonds in the different lipid classes. The separation was applied for a rapid monitoring of the lipid composition in thylakoids and in fractionated inner and outer envelopes. The activities of galactosyltransferases involved in galactolipid metabolism, UDPGal:diacylglycerol galactosyltransferase and galactolipid:galactolipid galactosyltransferase, could be measured quantitatively in specific assays for both enzymes.

Effects of frost hardening on the composition of galactolipids and phospholipids occurring during isolation of chloroplast thylakoids from needles of scots pine

Frost hardening of seedlings of Scots pine ( Pinus sylvestris) at a non-freezing temperature of 4°C resulted in a 2-fold increase of the acyl lipids of the needles. This was because of increases in phospholipids and triglycerides. The galactolipid content of the needles was almost the same in unhardened and frost-hardened seedlings. In unhardened seedlings the mol ratio of monogalactosyl diacylglycerol (MGDG) to digalactosyl diacylglycerol (DGDG) was 1.7 ± 0.3 and 0.9 ± 0.2 in needles and isolated thylakoids, respectively. Corresponding ratios for frost-hardened seedlings were 1.5 ± 0.2 and 0.3 ± 0.03. The lower ratios found in isolated thylakoids, particularly in thylakoids from frost-hardened seedlings, are suggested to depend on the enzyme galactolipid: galactolipid galactosyltransferase being active during the isolation procedure. This is deduced from the result that the content of MGDG decreased and that of DGDG and 1.2 diglycerides increased. Needles of Scots pine also contain phospholipidase D. This enzyme was active during thylakoid preparation, particularly after frost hardening, as judged from the large amount of phosphatidic acid found the in thylakoid fraction isolated from frost-hardening needles. The fatty acid composition of the acyl lipids showed no major changes due to hardening at non-freezing temperature.

Spinach chloroplasts: localization of enzymes involved in galactolipid metabolism

A shortened procedure for the separation of spinach chloroplast envelope membranes is presented, resulting in high yields of enriched inner and outer envelopes. Fractionated envelopes were tested for activities in galactolipid synthesis by measuring incorporation of UDPGal and by using a specific assay for galactolipid: galactolipid galactosyltransferase (GGGT); additionally, acyl-CoA synthetase activities were tested. UDPGal incorporation and GGGT were found predominantly in enriched inner envelopes, acyl-CoA synthetase was predominantly in enriched outer envelopes. These observations point to a localization of both UPDGal: diacylglycerol galactosyltransferase (UDGT) and GGGT in the inner membrane. Alternatively, chloroplasts were incubated with [ 14C]acetate under conditions permitting fatty acid synthesis in the presence and absence of UDP[ 3H]Gal. After incubation, envelope fractions were isolated and analyzed for labeled lipids. Analysis agreed with localization of UDGT in the inner envelope. However, labeled products of GGGT were found both in enriched inner and outer envelopes. Furthermore, inactivation of GGGT by the proteinase thermolysin indicates a location on the cytosolic face of the chloroplast. These apparently contradictory results concerning GGGT might be explained by the involvement of contact sites between inner and outer envelopes or by lipid transformation during membrane fractionation.

Turnover of galactolipids incorporated into chloroplast envelopes: An assay for galactolipid: Galactolipid galactosyltransferase

An assay for the measurement of galactolipid:galactolipid galactosyltransferase activity in chloroplast envelopes is presented. For this assay, [ 14C]galactose-labeled monogalactosyl diacylglycerol and sodium desoxycholate (in the molar ratio 1:10) are sonicated together, and the resulting suspension is resonicated with the chloroplast envelopes (in the molar ratio total galactolipids/sodium desoxycholate = 0.6). The incorporated monogalactosyl diacylglycerol is readily converted into di-, tri-, tetra- and, presumably, pentagalactosyl diacylglycerol. The galactosyltransferase activity shows a broad pH optimum of 5.9–7.0, it is still active at temperatures as low as 4°C and is stimulated by Mg 2+ and Mn 2+. The detergent-modified envelopes show a reduced incorporation of UDP[ 3H]Gal, which results, however, in a quite normal pattern of 3H-Iabeled galactolipids. The rate of conversion of the incorporated [ 14C]monogalactosyl diacylglycerol was not influenced by the presence or absence of UDP[ 3H]Gal. Evidence is supplied for the absence of UDPgalactose:monogalactosyl diacylglycerol galactosyltransferase activity in our system. Incorporation of [ 14C)digalactosyl diacylglycerol into the envelopes yields labeled tri- and tetragalactosyl diacylglycerol and also monogalactosyl diacylglycerol. The possibility of reversed galactolipid: galactolipid galactosyltransferase activity is discussed. Incorporated monogalactosyl monoacylglycerol is converted partly into a compound which is presumably digalactosyl monoacylglycerol.