Critical regulation of galactolipid synthesis controls membrane differentiation and remodeling in distinct plant organs and following environmental changes

The plant galactolipids, monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG), are the most abundant lipids in chloroplast membranes, and they constitute the majority of total membrane lipids in plants. MGDG is synthesized by two types of MGDG synthase, type-A (MGD1) and type-B (MGD2, MGD3). These MGDG synthases have distinct roles in Arabidopsis. In photosynthetic organs, Type A MGD is responsible for the bulk of MGDG synthesis, whereas Type B MGD is expressed in non-photosynthetic organs such as roots and flowers and mainly contributes to DGDG accumulation under phosphate deficiency. Similar to MGDG synthesis, DGDG is synthesized by two synthases, DGD1 and DGD2; DGD1 is responsible for the majority of DGDG synthesis, whereas DGD2 makes its main contribution under phosphate deficiency. These galactolipid synthases are regulated by light, plant hormones, redox state, phosphatidic acid levels, and various stress conditions such as drought and nutrient limitation. Maintaining the appropriate ratio of these two galactolipids in chloroplasts is important for stabilizing thylakoid membranes and maximizing the efficiency of photosynthesis. Here we review progress made in the last decade towards a better understanding of the pathways regulating plant galactolipid biosynthesis.     

Refolding from denatured inclusion bodies,purification to homogeneity and simplified assay of MGDG synthases from land plants

In plant cells, the synthesis of monogalactosyldiacylglycerol (MGDG) is catalyzed within plastid envelope membranes by MGD proteins. MGDG synthesis was also reported in apicomplexan parasites, a phylum of protists harbouring a plastid that proved essential for the parasite survival. MGD activity is therefore a potent target for herbicidal and anti-parasitic molecules. In this study, we describe a detailed in vitro refolding protocol for denatured recombinant MGD accumulated in inclusion bodies from transformed Escherichia coli. The refolding process was dependent on CHAPS detergent and lipids, such as diacylglycerol and phosphatidylglycerol, as well as bivalent metals. Owing to this refolding procedure, the recombinant MGD protein from spinach was purified to homogeneity, allowing a definite characterization of its non-processivity and an investigation of its dimerization using cross-linking reagents. Additionally, using the portion of recombinant enzyme that accumulates in an active form in bacterial membranes, we developed a miniature assay for high-throughput screening for inhibitors.     

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.     

Phylogeny of galactolipid synthase homologs together with their enzymatic analyses revealed a possible origin and divergence time for photosynthetic membrane biogenesis

The photosynthetic membranes of cyanobacteria and chloroplasts of higher plants have remarkably similar lipid compositions. In particular, thylakoid membranes of both cyanobacteria and chloroplasts are composed of galactolipids, of which monogalactosyldiacylglycerol (MGDG) is the most abundant, although MGDG biosynthetic pathways are different in these organisms. Comprehensive phylogenetic analysis revealed that MGDG synthase (MGD) homologs of filamentous anoxygenic phototrophs Chloroflexi have a close relationship with MGDs of Viridiplantae (green algae and land plants). Furthermore, analyses for the sugar specificity and anomeric configuration of the sugar head groups revealed that one of the MGD homologs exhibited a true MGDG synthetic activity. We therefore presumed that higher plant MGDs are derived from this ancestral type of MGD genes, and genes involved in membrane biogenesis and photosystems have been already functionally associated at least at the time of Chloroflexi divergence. As MGD gene duplication is an important event during plastid evolution, we also estimated the divergence time of type A and B MGDs. Our analysis indicated that these genes diverged -323 million years ago, when Spermatophyta (seed plants) were appearing. Galactolipid synthesis is required to produce photosynthetic membranes; based on MGD gene sequences and activities, we have proposed a novel evolutionary model that has increased our understanding of photosynthesis evolution.     

Structural insights and membrane binding properties of MGD1, the major galactolipid synthase in plants

Monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG) are the major lipid components of photosynthetic membranes, and hence the most abundant lipids in the biosphere. They are essential for assembly and function of the photosynthetic apparatus. In Arabidopsis, the first step of galactolipid synthesis is catalyzed by MGDG synthase 1 (MGD1), which transfers a galactosyl residue from UDP‐galactose to diacylglycerol (DAG). MGD1 is a monotopic protein that is embedded in the inner envelope membrane of chloroplasts. Once produced, MGDG is transferred to the outer envelope membrane, where DGDG synthesis occurs, and to thylakoids. Here we present two crystal structures of MGD1: one unliganded and one complexed with UDP. MGD1 has a long and flexible region (approximately 50 amino acids) that is required for DAG binding. The structures reveal critical features of the MGD1 catalytic mechanism and its membrane binding mode, tested on biomimetic Langmuir monolayers, giving insights into chloroplast membrane biogenesis. The structural plasticity of MGD1, ensuring very rapid capture and utilization of DAG, and its interaction with anionic lipids, possibly driving the construction of lipoproteic clusters, are consistent with the role of this enzyme, not only in expansion of the inner envelope membrane, but also in supplying MGDG to the outer envelope and nascent thylakoid membranes.     

Type A and type B monogalactosyldiacylglycerol synthases are spatially and functionally separated in the plastids of higher plants

Mono- and digalactosyldiacylglycerol (MGDG and DGDG, respectively) constitute the bulk of membrane lipids in plant chloroplasts. The final step in MGDG biosynthesis occurs in the plastid envelope and is catalyzed by MGDG synthase. In Arabidopsis, the three MGDG synthases are classified into type A (atMGD1) and type B MGD isoforms (atMGD2 and atMGD3). atMGD1 is an inner envelope membrane-associated protein of chloroplasts and is responsible for the bulk of galactolipid biosynthesis in green tissues. MGD1 function is indispensable for thylakoid membrane biogenesis and embryogenesis. By contrast, type B atMGD2 and atMGD3 are localized in the outer envelopes and have no important role in chloroplast biogenesis or plant development under nutrient-sufficient conditions. These type B MGD genes are, however, strongly induced by phosphate (Pi) starvation and are essential for alternative galactolipid biosynthesis during Pi starvation. MGD1 gene expression is up-regulated by light and cytokinins. By contrast, Pi starvation-dependent expression of atMGD2/3 is suppressed by cytokinins but induced through auxin signaling pathways. These growth factors may control the functional sharing of the inner envelope pathway by atMGD1 and the outer envelope pathway by atMGD2/3 according to the growth environment.     

Molecular modeling and site-directed mutagenesis of plant chloroplast monogalactosyldiacylglycerol synthase reveal critical residues for activity

Monogalactosyldiacylglycerol (MGDG), the major lipid of plant and algal plastids, is synthesized by MGD (or MGDG synthase), a dimeric and membrane-bound glycosyltransferase of the plastid envelope that catalyzes the transfer of a galactosyl group from a UDP-galactose donor onto a diacylglycerol acceptor. Although this enzyme is essential for biogenesis, and therefore an interesting target for herbicide design, no structural information is available. MGD monomers share sequence similarity with MURG, a bacterial glycosyltransferase catalyzing the transfer of N-acetyl-glucosamine on Lipid 1. Using the x-ray structure of Escherichia coli MURG as a template, we computed a model for the fold of Spinacia oleracea MGD. This structural prediction was supported by site-directed mutagenesis analyses. The predicted monomer architecture is a double Rossmann fold. The binding site for UDP-galactose was predicted in the cleft separating the two Rossmann folds. Two short segments of MGD (beta2-alpha2 and beta6-beta7 loops) have no counterparts in MURG, and their structure could not be determined. Combining the obtained model with phylogenetic and biochemical information, we collected evidence supporting the beta2-alpha2 loop in the N-domain as likely to be involved in diacylglycerol binding. Additionally, the monotopic insertion of MGD in one membrane leaflet of the plastid envelope occurs very likely at the level of hydrophobic amino acids of the N-terminal domain.     

Role of galactolipid biosynthesis in coordinated development of photosynthetic complexes and thylakoid membranes during chloroplast biogenesis in Arabidopsis

The galactolipids monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG) are the predominant lipids in thylakoid membranes and indispensable for photosynthesis. Among the three isoforms that catalyze MGDG synthesis in Arabidopsis thaliana, MGD1 is responsible for most galactolipid synthesis in chloroplasts, whereas MGD2 and MGD3 are required for DGDG accumulation during phosphate (Pi) starvation. A null mutant of Arabidopsis MGD1 (mgd1‐2), which lacks both galactolipids and shows a severe defect in chloroplast biogenesis under nutrient‐sufficient conditions, accumulated large amounts of DGDG, with a strong induction of MGD2/3 expression, during Pi starvation. In plastids of Pi‐starved mgd1‐2 leaves, biogenesis of thylakoid‐like internal membranes, occasionally associated with invagination of the inner envelope, was observed, together with chlorophyll accumulation. Moreover, the mutant accumulated photosynthetic membrane proteins upon Pi starvation, indicating a compensation for MGD1 deficiency by Pi stress‐induced galactolipid biosynthesis. However, photosynthetic activity in the mutant was still abolished, and light‐harvesting/photosystem core complexes were improperly formed, suggesting a requirement for MGDG for proper assembly of these complexes. During Pi starvation, distribution of plastid nucleoids changed concomitantly with internal membrane biogenesis in the mgd1‐2 mutant. Moreover, the reduced expression of nuclear‐ and plastid‐encoded photosynthetic genes observed in the mgd1‐2 mutant under Pi‐sufficient conditions was restored after Pi starvation. In contrast, Pi starvation had no such positive effects in mutants lacking chlorophyll biosynthesis. These observations demonstrate that galactolipid biosynthesis and subsequent membrane biogenesis inside the plastid strongly influence nucleoid distribution and the expression of both plastid‐ and nuclear‐encoded photosynthetic genes, independently of photosynthesis.     

Type-B monogalactosyldiacylglycerol synthases are involved in phosphate starvation-induced lipid remodeling, and are crucial for low-phosphate adaptation

Mono- and digalactosyldiacylglycerol (MGDG and DGDG, respectively) constitute the bulk of membrane lipids in plant chloroplasts. Mutant analyses in Arabidopsis have shown that these galactolipids are essential for chloroplast biogenesis and photoautotrophic growth. Moreover, these non-phosphorous lipids are proposed to participate in low-phosphate (Pi) adaptations. Under Pi-limited conditions, a drastic accumulation of DGDG occurs concomitantly with a large reduction in membrane phospholipids, suggesting that plants substitute DGDG for phospholipids during Pi starvation. Previously, we reported that among the three MGDG synthase genes ( MGD1 , MGD2 and MGD3 ), the type-B MGD2 and MGD3 are upregulated in parallel with DGDG synthase genes during Pi starvation. Here, we describe the identification and characterization of T-DNA insertional mutants of Arabidopsis type-B MGD genes. Under Pi-starved conditions, the mgd3-1 mutant showed a drastic reduction in DGDG accumulation, particularly in the root, indicating that MGD3 is the main isoform responsible for DGDG biosynthesis in Pi-starved roots. Moreover, in the roots of mgd2 mgd3 plants, Pi stress-induced accumulation of DGDG was almost fully abolished, showing that type-B MGD enzymes are essential for membrane lipid remodeling in Pi-starved roots. Reductions in fresh weight, root growth and photosynthetic performance were also observed in these mutants under Pi-starved conditions. These results demonstrate that Pi stress-induced membrane lipid remodeling is important in plant growth during Pi starvation. The widespread distribution of type-B MGD genes in land plants suggests that membrane lipid remodeling mediated by type-B MGD enzymes is a potent adaptation to Pi deficiency for land plants.     

Revisiting the expression and purification of MGD1, the major galactolipid synthase in Arabidopsis to establish a novel standard for biochemical and structural studies

Monogalactosyldiacylglycerol, the major lipid of plants and algal plastids, is synthesized by MGDG synthases (MGD). MGDs belong to the large glycosyltransferase family. They catalyze the transfer of a galactose residue from the donor UDP-Gal to a 1,2-sn-diacylglycerol acceptor. MGDs are monotopic proteins localized in the plastid envelope and, as such, they are difficult to purify. This study re-examined previous purification procedures and aimed to set up a standard protocol for expression and purification of recombinant MGD1, addressing problems frequently encountered with the purification of glycosyltransferases, particularly protein aggregation, and enabling crystallization for structural studies. Briefly, His-tagged versions of MGD1 were expressed in Escherichia coli and purified by a two-step procedure, including immobilized metal affinity chromatography and size-exclusion chromatography. We demonstrated that E. coli is an appropriate host cell to produce a soluble and active form of MGD1. We also investigated the effects of various buffers and additives used during the purification and concentration steps on the biochemical behavior of the enzyme. The protocol we developed typically yields milligram quantities of pure and homogenous protein material and proved suitable for crystallization and biochemical studies. We also revisited the conditions for activity tests and effects of known positive effectors of MGD1 such as phosphatidic acid and phosphatidylglycerol.     

A predicted plastid rhomboid protease affects phosphatidic acid metabolism in Arabidopsis thaliana

The thylakoid membranes of the chloroplast harbor the photosynthetic machinery that converts light into chemical energy. Chloroplast membranes are unique in their lipid makeup, which is dominated by the galactolipids mono‐ and digalactosyldiacylglycerol (MGDG and DGDG). The most abundant galactolipid, MGDG, is assembled through both plastid and endoplasmic reticulum (ER) pathways in Arabidopsis, resulting in distinguishable molecular lipid species. Phosphatidic acid (PA) is the first glycerolipid formed by the plastid galactolipid biosynthetic pathway. It is converted to substrate diacylglycerol (DAG) for MGDG Synthase (MGD1) which adds to it a galactose from UDP‐Gal. The enzymatic reactions yielding these galactolipids have been well established. However, auxiliary or regulatory factors are largely unknown. We identified a predicted rhomboid‐like protease 10 (RBL10), located in plastids of Arabidopsis thaliana, that affects galactolipid biosynthesis likely through intramembrane proteolysis. Plants with T‐DNA disruptions in RBL10 have greatly decreased 16:3 (acyl carbons:double bonds) and increased 18:3 acyl chain abundance in MGDG of leaves. Additionally, rbl10‐1 mutants show reduced [¹⁴C]–acetate incorporation into MGDG during pulse?chase labeling, indicating a reduced flux through the plastid galactolipid biosynthesis pathway. While plastid MGDG biosynthesis is blocked in rbl10‐1 mutants, they are capable of synthesizing PA, as well as producing normal amounts of MGDG by compensating with ER‐derived lipid precursors. These findings link this predicted protease to the utilization of PA for plastid galactolipid biosynthesis potentially revealing a regulatory mechanism in chloroplasts.     

Oxidative stress and thioredoxin-interacting protein promote intravasation of melanoma cells

Although intravasation may be a critical rate-limiting step in the metastatic cascade, the role of oxidative stress in intravasation is unknown. We tested the hypothesis that reactive oxygen species (ROS), regulated by thioredoxin interacting protein (Txnip) through the action of thioredoxin (Trx), influence human SK-MEL-28 melanoma cell reverse (basolateral-to-apical) transendothelial migration (TEM) in vitro as a model for intravasation. Reverse transendothelial migration was dose-dependently induced by hydrogen peroxide 2.4-fold for 0.1 microM (P < 0.01) and 3.9-fold for 1 microM (P < 0.001) vs. control, and this effect was blocked by the antioxidant N-acetylcysteine. Overexpression of Txnip by infecting melanoma cells with adenovirus increased TEM 3-fold vs. control (P < 0.001), and this increase was blocked by N-acetylcysteine, indicating a redox-sensitive mechanism. Conversely, thioredoxin overexpression blocked hydrogen peroxide-induced TEM. Exposure to ultraviolet-A radiation increased ROS 1.8-fold (P < 0.01), and this was accompanied by a 45% reduction (P < 0.05) in thioredoxin activity and an 11.4-fold (P < 0.001) increase in Txnip gene expression. These data suggest that TEM of melanoma cells during intravasation is in part mediated by ROS-sensitive cellular signaling cascades, may be controlled by Txnip and its interaction with thioredoxin that in turn modulates cellular levels of oxidative stress, and may be initiated by ultraviolet-A induction of this cascade.     

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.     

A Monogalactosyldiacylglycerol Synthase Found in the Green Sulfur Bacterium Chlorobaculum tepidum Reveals Important Roles for Galactolipids in Photosynthesis

Monogalactosyldiacylglycerol (MGDG), which is conserved in almost all photosynthetic organisms, is the most abundant natural polar lipid on Earth. In plants, MGDG is highly accumulated in the chloroplast membranes and is an important bulk constituent of thylakoid membranes. However, precise functions of MGDG in photosynthesis have not been well understood. Here, we report a novel MGDG synthase from the green sulfur bacterium Chlorobaculum tepidum. This enzyme, MgdA, catalyzes MGDG synthesis using UDP-Gal as a substrate. The gene encoding MgdA was essential for this bacterium; only heterozygous mgdA mutants could be isolated. An mgdA knockdown mutation affected in vivo assembly of bacteriochlorophyll c aggregates, suggesting the involvement of MGDG in the construction of the light-harvesting complex called chlorosome. These results indicate that MGDG biosynthesis has been independently established in each photosynthetic organism to perform photosynthesis under different environmental conditions. We complemented an Arabidopsis thaliana MGDG synthase mutant by heterologous expression of MgdA. The complemented plants showed almost normal levels of MGDG, although they also had abnormal morphological phenotypes, including reduced chlorophyll content, no apical dominance in shoot growth, atypical flower development, and infertility. These observations provide new insights regarding the importance of regulated MGDG synthesis in the physiology of higher plants.     

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.     

The thioredoxin system of the filamentous fungus Aspergillus nidulans: impact on development and oxidative stress response

Redox regulation has been shown to be of increasing importance for many cellular processes. Here, redox homeostasis was addressed in Aspergillus nidulans, an important model organism for fundamental biological questions such as development, gene regulation or the regulation of the production of secondary metabolites. We describe the characterization of a thioredoxin system from the filamentous fungus A. nidulans. The A. nidulans thioredoxin A (AnTrxA) is an 11.6-kDa protein with a characteristic thioredoxin active site motif (WCGPC) encoded by the trxA gene. The corresponding thioredoxin reductase (AnTrxR), encoded by the trxR gene, represents a homodimeric flavoprotein with a native molecular mass of 72.2 kDa. When combined in vitro, the in Escherichia coli overproduced recombinant proteins AnTrxA and AnTrxR were able to reduce insulin and oxidized glutathione in an NADPH-dependent manner indicating that this in vitro redox system is functional. Moreover, we have created a thioredoxin A deletion strain that shows decreased growth, an increased catalase activity, and the inability to form reproductive structures like conidiophores or cleistothecia when cultivated under standard conditions. However, addition of GSH at low concentrations led to the development of sexual cleistothecia, whereas high GSH levels resulted in the formation of asexual conidiophores. Furthermore, by applying the principle of thioredoxin-affinity chromatography we identified several novel putative targets of thioredoxin A, including a hypothetical protein with peroxidase activity and an aldehyde dehydrogenase.