Monogalactosyldiacylglycerol Synthase Research Articles

Drought stress affects chloroplast lipid metabolism in rape (Brassica napus) leaves.

Rape (Brassica napus L. var. Bienvenue) is a 16:3 plant which contains predominantly prokaryotic species of monogalactosyldiacylglycerol i.e. sn-1 C18, sn-2 C16 (C18/C16 MGDG). Rape plants were exposed to a restricted water supply for 12 days. Under drought conditions, considerable changes in lipid metabolism were observed. Drought stress provoked a decline in leaf polar lipids, which is mainly due to a decrease in MGDG content. Determination of molecular species in phosphatidylcholine (PC) and MGDG indicated that the prokaryotic molecular species of MGDG (C18/C16) decreased after drought stress while the eukaryotic molecular species (C18/C18) remained stable. Drought stress had different effects on two key enzymes of PC and MGDG synthesis. The in vitro activity of MGDG synthase (EC. 2.4.1.46) was reduced in drought stressed plants whereas cholinephosphotransferase (EC. 2.7.8.2) activity was not affected. Altogether these results suggest that the prokaryotic pathway leading to MGDG synthesis was strongly affected by drought stress while the eukaryotic pathway was not. It was also observed that the molecular species of leaf PC became more saturated in drought stressed plants. This could be due to a specific decrease in oleate desaturase activity.

Two types of MGDG synthase genes, found widely in both 16:3 and 18:3 plants, differentially mediate galactolipid syntheses in photosynthetic and nonphotosynthetic tissues in Arabidopsis thaliana.

In Arabidopsis, monogalactosyldiacylglycerol (MGDG) is synthesized by a multigenic family of MGDG synthases consisting of two types of enzymes differing in their N-terminal portion: type A (atMGD1) and type B (atMGD2 and atMGD3). The present paper compares type B isoforms with the enzymes of type A that are known to sit in the inner membrane of plastid envelope. The occurrence of types A and B in 16:3 and 18:3 plants shows that both types are not specialized isoforms for the prokaryotic and eukaryotic glycerolipid biosynthetic pathways. Type A atMGD1 gene is abundantly expressed in green tissues and along plant development and encodes the most active enzyme. Its mature polypeptide is immunodetected in the envelope of chloroplasts from Arabidopsis leaves after cleavage of its transit peptide. atMGD1 is therefore likely devoted to the massive production of MGDG required to expand the inner envelope membrane and build up the thylakoids network. Transient expression of green fluorescent protein fusions in Arabidopsis leaves and in vitro import experiments show that type B precursors are targeted to plastids, owing to a different mechanism. Noncanonical addressing peptides, whose processing could not be assessed, are involved in the targeting of type B precursors, possibly to the outer envelope membrane where they might contribute to membrane expansion. Expression of type B enzymes was higher in nongreen tissues, i.e., in inflorescence (atMGD2) and roots (atMGD3), where they conceivably influence the eukaryotic structure prominence in MGDG. In addition, their expression of type B enzymes is enhanced under phosphate deprivation.

The Digalactosyldiacylglycerol (DGDG) Synthase DGD1 Is Inserted into the Outer Envelope Membrane of Chloroplasts in a Manner Independent of the General Import Pathway and Does Not Depend on Direct Interaction with Monogalactosyldiacylglycerol Synthase for DGDG Biosynthesis

Galactolipids make up the bulk of chloroplast lipids. Therefore, the genes involved in the synthesis of the galactolipids monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG) play a critical role in chloroplast development. In this study, we analyzed the subcellular localization of the Arabidopsis DGDG synthase DGD1, which was recently identified by complementation of the Arabidopsis dgd1 mutant. In vitro import experiments demonstrated that DGD1 was targeted to the chloroplast outer envelope in an ATP-independent manner. DGD1 could not be extracted from the membranes by high salt or alkali, suggesting that it is an integral membrane protein. Uptake experiments with truncated versions of DGD1 indicated that the information for targeting and insertion into the outer envelope resides in the N-terminal half of DGD1, but not in the first 33 amino acids. DGD1 apparently does not contain a cleavable signal peptide. Antibodies to Arabidopsis DGD1 detected a 90-kDa protein localized to the chloroplast envelopes of both pea and Arabidopsis. Transformation of DGD1 constructs into cyanobacteria resulted in the expression of active DGDG synthase and demonstrated that DGDG synthesis depends on MGDG lipid, but does not require direct interaction with the plant MGDG synthase.

The multigenic family of monogalactosyl diacylglycerol synthases

Because the synthesis of monogalactosyldiacylglycerol (MGDG) is unique to plants, identified as an important marker of the plastid envelope, involved in a key step of plastid biogenesis and is the most abundant lipid on earth, MGDG synthase activity was extensively analysed at the biochemical and physiological levels. In the present paper, we present our current knowledge on the MGDG synthase's function, structure and topology in envelope membranes, and discuss possible roles in plant cell glycerolipid metabolism. The recent discovery of a multigenic family of MGDG synthases raised the possibility that multiple isoenzymes might carry out MGDG synthesis in various tissues and developmental stages.

Transcriptional regulation by light and phytohormones of the MGD gene in cucumber

Monogalactosyldiacylglycerol (MGDG) synthase catalyses formation of MGDG, a major structural lipid of chloroplasts. We have already cloned a cDNA for the synthase from a cucumber cDNA library and shown that expression of this gene is regulated by light and a phytohormone, cytokinin. In the present study, we report the molecular basis for transcriptional regulation by light and cytokinin in detail. First, in terms of the enzyme activity, gradual increases in activity mediated by light and cytokinin treatments were observed. At the same time, however, the changes in the mRNA level showed different profiles, with a transient peak during the early stages of light and cytokinin treatment. The interval between the peak level of mRNA and enzyme activation implies the existence of a post-transcriptional regulatory system. In addition, a genomic clone of MGDG synthase isolated from a cucumber genome library was used for a motif search in databases, and this revealed that putative cis-acting elements for light and phytohormones exist in the 5Î-upstream region of the MGD cucumber gene. Detailed analysis of this region for light- and cytokinin-responsive activity was performed using a – 90 truncated 35 S minimal promoter/luciferase (LUC) reporter gene. It resulted in high levels of LUC expression in etiolated cucumber cotyledons in response to illumination and cytokinin treatment. These results indicate that this 5Î-upstream region is involved in light- and cytokinin-enhanced MGD gene expression, and that light- and cytokinin-responsive enhancements of the MGDG synthase activity are regulated, at least in part, at the level of transcription.

Transcriptional regulation by light and phytohormones of the MGD gene in cucumber

Monogalactosyldiacylglycerol (MGDG) synthase catalyses formation of MGDG, a major structural lipid of chloroplasts. We have already cloned a cDNA for the synthase from a cucumber cDNA library and shown that expression of this gene is regulated by light and a phytohormone, cytokinin. In the present study, we report the molecular basis for transcriptional regulation by light and cytokinin in detail. First, in terms of the enzyme activity, gradual increases in activity mediated by light and cytokinin treatments were observed. At the same time, however, the changes in the mRNA level showed different profiles, with a transient peak during the early stages of light and cytokinin treatment. The interval between the peak level of mRNA and enzyme activation implies the existence of a post-transcriptional regulatory system. In addition, a genomic clone of MGDG synthase isolated from a cucumber genome library was used for a motif search in databases, and this revealed that putative cis-acting elements for light and phytohormones exist in the 5'-upstream region of the MGD cucumber gene. Detailed analysis of this region for light- and cytokinin-responsive activity was performed using a -90 truncated 35 S minimal promoter/luciferase (LUC) reporter gene. It resulted in high levels of LUC expression in etiolated cucumber cotyledons in response to illumination and cytokinin treatment. These results indicate that this 5'-upstream region is involved in light- and cytokinin-enhanced MGD gene expression, and that light- and cytokinin-responsive enhancements of the MGDG synthase activity are regulated, at least in part, at the level of transcription.

The multigenic family of monogalactosyl diacylglycerol synthases

The multigenic family of monogalactosyl diacylglycerol synthases

Biochemical and topological properties of type A MGDG synthase, a spinach chloroplast envelope enzyme catalyzing the synthesis of both prokaryotic and eukaryotic MGDG.

MGDG synthase, the enzyme that catalyzes the synthesis of the major chloroplast membrane lipid monogalactosyldiacylglycerol (MGDG), is encoded by a multigenic family. We have analyzed the biochemical properties, subcellular localization and membrane topology of a spinach chloroplast MGDG synthase, a representative member of the type A family from Spinacia oleracea (soMGD A), using a recombinant protein that was functionally overexpressed in Escherichia coli and specific polyclonal antibodies. We demonstrated that soMGD A could catalyze the synthesis of both 'prokaryotic' and 'eukaryotic' MGDG molecular species in vitro, with a selectivity for diacylglycerol similar to that of purified chloroplast envelope MGDG synthase activity. Furthermore, soMGD A was shown to be sensitive to chemical reagents (dithiothreitol, N-ethylmaleimide and o-phenanthroline) known to affect MGDG synthesis by the partially purified enzyme, as well as in isolated chloroplast envelope membranes. In spinach chloroplasts, soMGD A was localized by Western blot analysis in the inner envelope membrane. Topological studies demonstrated that soMGD A is a monotopic enzyme, embedded within one leaflet of the inner envelope membrane from spinach chloroplasts, a structure which may involve amphipathic alpha helices. We further demonstrated that in vitro, soMGD A precursor is imported and processed to its correct mature form in intact chloroplasts. These results show that soMGD A corresponds to a mature polypeptide of approximately 45 kDa. In addition, inactivation kinetics after gamma-ray irradiation strongly suggest that both native chloroplast envelope MGDG synthase and recombinant soMGD A have a functional molecular mass of 95-100 kDa, indicating that they are probably active as homodimers made of two 45-kDa subunits. This study suggests that, in spite of the growing evidence that MGDG synthesis is catalyzed by a multigenic family of enzymes, in spinach leaves both prokaryotic and eukaryotic MGDG syntheses could be attributable to a unique dimeric enzyme, provided that diacylglycerol is transported from the outer membrane to the inner membrane of the chloroplast envelope.

A Bacillus subtilis gene encoding a protein similar to nucleotide sugar transferases influences cell shape and viability.

Bacillus subtilis gene ypfP, which is located at 196 degrees on the genetic map, shows similarity to both the monogalactosyldiacylglycerol synthase gene of Cucumis sativus, which encodes a galactosyltransferase, and the murG genes of B. subtilis, Escherichia coli, Haemophilus influenzae, and Synechocystis sp. strain PCC6803, which encode N-acetylglucosaminyltransferases involved in peptidoglycan biosynthesis. Cells containing a null mutation of ypfP are shorter and rounder than wild-type cells during growth in Luria-Bertani medium and glucose minimal medium. In addition, the mutant cells preferentially undergo lysis when grown on solid Luria-Bertani medium.

The Catalytic Site of Monogalactosyldiacylglycerol Synthase from Spinach Chloroplast Envelope Membranes: BIOCHEMICAL ANALYSIS OF THE STRUCTURE AND OF THE METAL CONTENT

We have analyzed the structure of the active site of monogalactosyldiacylglycerol (MGDG) synthase from spinach chloroplast envelope. Since purification of this membrane-embedded enzyme yielded such low amounts of protein that analyses of the amino acid sequence were so far impossible, we used indirect strategies. Analyses of the inhibition of MGDG synthase by UDP and of its inactivation by citraconic anhydride first indicated that the enzyme contained two functionally independent and topologically distinct binding sites for each substrate. Whereas MGDG synthase binds both the nucleotidic part of UDP-Gal and the acyl chains of 1,2-diacylglycerol, UDP is a competitive inhibitor relatively to UDP-Gal, while it does not compete with 1,2-diacylglycerol for binding on the enzyme. The UDP-Gal-binding site contains lysine residues, as demonstrated for UDP-Gal-binding sites from all galactosyltransferases studied so far. Radiolabeling of MGDG synthase by sulfur labeling reagent, a 35S-labeled lysine-blocking reagent, confirmed that MGDG synthase was a polypeptide with a low molecular mass (around 20 kDa). The 1,2-diacylglycerol-binding site contains reduced cysteines and at least one metal. The divalent cation(s) associated to apo-MGDG synthase was not unambiguously identified, but the results suggest that it could be zinc. Therefore, MGDG synthase presents some structural features in common with diacylglycerol-manipulating enzymes, such as protein kinase C and 1,2-diacylglycerol kinase, which are characterized by the presence of a ubiquitous Cys6His2 domain involved in zinc coordination in their 1,2-diacylglycerol-binding domains.

Comparison of the kinetic properties of MGDG synthase in mixed micelles and in envelope membranes from spinach chloroplast

We have applied the ‘membrane partition’ kinetic modelling approach proposed by Heirwegh et al. [(1988)] Biochem. J. 254, 101–108] to MGDG synthase in isolated envelope vesicles. Comparison of the kinetic parameters obtained for MGDG synthase assayed in purified envelope membranes and in mixed-micelles demonstrates that the latter are relevant to the situation in envelope membranes and that MGDG synthase has a very high affinity for dilinoleoylglycerol. Our results provide additional evidence for the hypothesis that the high affinity of the envelope MGDG synthase for dilinoleoylglycerol could be responsible for the presence of C18 fatty acids at both the sn-1 and sn-2 position of the glycerol backbone in MGDG.

Kinetic properties of monogalactosyldiacylglycerol synthase from spinach chloroplast envelope membranes.

We have investigated the functioning of monogalactosyldiacylglycerol (MGDG) synthase activity partially purified from spinach chloroplast envelope membranes, using mixed micelles containing diacylglycerol (the substrate for MGDG synthase), CHAPS (3-[(cholamidopropyl)dimethylammonio]-1- propanesulfonic acid), and phosphatidylglycerol. The presence of this anionic phospholipid was essential for optimal MGDG synthase activity because it strongly improves diacylglycerol solubilization by CHAPS. We have demonstrated that the "surface dilution" kinetic model proposed by Deems et al. (Deems, R.A., Eaton, B.R., and Dennis, E.A. (1975) J. Biol. Chem. 250, 9013-9020) is valid for MGDG synthase assayed in mixed micelles within a narrow range of CHAPS concentration. However, the experimental conditions we have set up in this study led to the description of defined equilibrium and kinetic parameters of the interaction of the envelope MGDG synthase with diacylglycerol. Two-substrate kinetic studies were performed with varied UDP-galactose molar concentrations and varied dioleoylglycerol surface concentrations. The families of reciprocal plots obtained were shown to intersect at a single point of the 1/[substrate] axis thus demonstrating that MGDG synthase is a sequential, either random or ordered, bireactant system. Therefore, MGDG synthase possesses two distinct and independent substrate-binding sites, a hydrophilic one for UDP-galactose and a hydrophobic one for diacylglycerol. The dependence of kinetic parameters on the diacylglycerol mol fraction allows a comparison of the affinity of the enzyme for a wide range of diacylglycerol molecular species. The Km values obtained were ranging between 0.0089 mol fraction (52 microM) for dilinoleoylglycerol (18:2/18:2) to 0.0666 mol fraction (416 microM) for distearoylglycerol (18:0/18:0), but the differences observed were not really related to the unsaturation of the molecule since the Km value for dilinoleoylglycerol was much lower than that (0.040 mol fraction) for dilinoleoylglycerol (18:3/18:3). The Km values for dioleoylglycerol (18:1/18:1) and for the diacylglycerol molecular species synthesized within chloroplasts, i.e. containing 18:1/16:0, were in the average range, i.e. lower than 0.030 mol fraction (around 170 microM).

Do thylakoids really contain phosphatidylcholine?

Isolated intact spinach chloroplasts were incubated with phospholipase C (phosphatidylcholine cholinephosphohydrolase, EC 3.1.4.3) under mild experimental conditions in which only the phosphatidylcholine localized in the cytosolic leaflet of the outer envelope membrane can be hydrolyzed. Thylakoids, which were protected from phospholipase C degradation, were subsequently prepared from the phospholipase C-treated chloroplasts and found to be devoid of phosphatidylcholine. Previously reported occurrences of phosphatidylcholine in thylakoid preparations probably reflect contamination of the thylakoids by envelope membranes. In the present work, contamination of thylakoids by envelope membranes was determined by measuring the 1,2-diacylglycerol 3-beta-galactosyltransferase [monogalactosyldiacylglycerol (MGDG) synthase; UDPgalactose: 1,2-diacylglycerol 3-beta-D-galactosyltransferase, EC 2.4.1.46] in the different chloroplast subfractions. We conclude that phosphatidylcholine is not present in highly purified thylakoids. Phosphatidylcholine is also absent from prokaryotic cyanobacterial membranes, and our results are in agreement with the endosymbiotic origin of higher plant chloroplasts.

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.

Lipid requirement and kinetic studies of solubilized UDP-galactose:diacylglycerol galactosyltransferase activity from spinach chloroplast envelope membranes

We have demonstrated a lipid requirement for the UDPgalactose:1,2-diacylglycerol 3-beta-D-galactosyl-transferase (or monogalactosyldiacylglycerol synthase; EC 2.4.1.46), an enzyme involved in the biosynthesis of monogalactosyldiacylglycerol, solubilized from chloroplast envelope membranes and partially purified by hydroxyapatite chromatography. The enzyme fraction was highly delipidated (<0.1 mg of lipid per mg of protein), and addition of lipids extracted from chloroplast membranes was necessary to reveal the activity. Acidic glycerolipids, and especially phosphatidylglycerol, were the best activators of the enzyme. The preparation of a delipidated enzyme fraction and the development of optimal assay conditions were prerequisites for the determination of the kinetic parameters for the hydrophobic substrate of the enzyme, diacylglycerol. In addition, we have demonstrated the existence of two substrate-binding sites: a hydrophobic one for diacylglycerol and a hydrophilic one for UDP-galactose.