[Show abstract][Hide abstract] ABSTRACT: The metabolome of an organism results from a complex biochemical network of thousands of enzymatic reaction and metabolites. Mass spectrometry (MS) in combination with extraction and separation methods has been successfully applied for the detection, identification, and quantitation of these chemical species. However, these methods are slow and provide limited information on the metabolic fluxes essential for the understanding of disease response to treatment and for mechanism of action studies. Stable isotope pulse-chase analysis can be used to study critical pathways in metabolic networks by monitoring the propagation of isotope tracers using MS.1 Pulse-chase analysis has been used in the clinical laboratory to determine the turnover rates of peptides in diseases such as amyloidosis. In the pulse phase of these experiments, cells assimilate the labeled molecules, whereas in the chase phase unlabeled molecules are reintroduced into the cells. Molecular turnover rates and half-lives of the affected molecules can be calculated by measuring the kinetics of these processes.
Mass Spectrometry Applications to the Clinical Lab, San Diego, CA; 03/2015
[Show abstract][Hide abstract] ABSTRACT: The diatom Phaeodactylum is rich in very long chain polyunsaturated fatty acids (PUFAs). Fatty acid (FA) synthesis, elongation, and desaturation have been studied in depth in plants including Arabidopsis, but for secondary endosymbionts the full picture remains unclear. FAs are synthesized up to a chain length of 18 carbons inside chloroplasts, where they can be incorporated into glycerolipids. They are also exported to the ER for phospho- and betaine lipid syntheses. Elongation of FAs up to 22 carbons occurs in the ER. PUFAs can be reimported into plastids to serve as precursors for glycerolipids. In both organelles, FA desaturases are present, introducing double bonds between carbon atoms and giving rise to a variety of molecular species. In addition to the four desaturases characterized in Phaeodactylum (FAD2, FAD6, PtD5, PtD6), we identified eight putative desaturase genes. Combining subcellular localization predictions and comparisons with desaturases from other organisms like Arabidopsis, we propose a scheme at the whole cell level, including features that are likely specific to secondary endosymbionts.
[Show abstract][Hide abstract] ABSTRACT: ] The pollen tube, a fast tip-growing cell, is an excellent model to study membrane and cell wall biosynthesis. Here, we describe a simple protocol using an easy to use device to perform immunofluorescence labelling of pollen tube membrane and cell wall. The use of the NucleoSpin column to perform all the steps of the immunolabelling procedure results in obtaining more intact pollen tubes.
[Show abstract][Hide abstract] ABSTRACT: Inorganic phosphate (Pi) is present in most soils at sub-optimal concentrations, strongly limiting plant development. Plants have the ability to sense and adapt to the surrounding ionic environment, and several genes involved in the response to Pi starvation have been identified. However, a global understanding of the regulatory mechanisms involved in this process is still elusive. Here, we have initiated a "chemical genetics" approach and isolated compounds that inhibit the response to Pi starvation in Arabidopsis thaliana. Molecules were screened for their ability to inhibit the expression of a Pi starvation marker gene (the high-affinity Pi transporter PHT1;4). A drug family named Phosphatin (Phosphate starvation inhibitor: PTN) was thus identified, whose members act as partial suppressors of Pi starvation responses. PTN addition also reduced various traits of Pi starvation such as phospholipid/glycolipid conversion, and the accumulation of starch and anthocyanins. A transcriptomic assay revealed a broad impact of PTN on the expression of many genes regulated by low Pi availability. Despite the reduced amount of Pi transporters and resulting reduced Pi uptake capacity, no reduction of Pi content was observed. In addition, PTN improved plant growth; this reveals that the developmental restrictions induced by Pi starvation are not a consequence of metabolic limitation, but also result from genetic regulation. This highlights the existence of signal transduction pathway(s) that limit plant development under the Pi starvation condition.
[Show abstract][Hide abstract] ABSTRACT: The metabolic profiling of various microalga species and their genetic variants, grown under varied environmental conditions, has become critical to accelerate the exploration of phytoplankton biodiversity and biology. The accumulation of valuable metabolites, such as glycerolipids, is also sought in microalgae for biotechnological applications ranging from food, feed, medicine, cosmetics to bioenergy and green chemistry. In this report we describe the direct analysis of metabolites and glycerolipids in small cell populations of the green alga Chlamydomonas reinhardtii, using laser ablation electrospray ionization (LAESI) mass spectrometry (MS) coupled with ion mobility separation (IMS). These microorganisms are capable of redirecting energy storage pathways from starch to neutral lipids depending on environmental conditions and nutrient availability. Metabolite and lipid production was monitored in wild type (WT), and genetically modified C. reinhardtii strains with an impaired starch pathway. Lipids, such as triacylglycerols (TAG) and diacylglyceryl-N,N,N-trimethylhomoserine (DGTS) were monitored over time under altered light conditions. More than 200 ions related to metabolites, e.g., arginine, cysteine, serine, palmitate, chlorophyll a, chlorophyll b, etc., were detected. The lipid profiles at different light intensities for strains with impaired starch pathway (Sta1 and Sta6) contained 26 glycerolipids, such as DGTS, monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG), as well as 33 TAG species. Results were obtained over a 72-hour time period in high and low light conditions for the WT species and the two mutants. Our results indicate that LAESI-IMS-MS can be utilized for the rapid analysis of increased TAG production at elevated light intensities. Compared to WT, the Sta6 strain showed 2.5-times higher lipid production at 72 hours under high light conditions. The results demonstrate our ability to rapidly observe numerous changes in metabolite and lipid levels in microalgae populations. These capabilities are expected to facilitate the exploration of genetically altered microalgae strains for biofuel production.
The Analyst 09/2014; 139(22). DOI:10.1039/C4AN01368A · 3.91 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In Angiosperms, the biosynthesis of galactolipids involves enzymes localized in the inner envelope membrane (IEM) of chloroplasts, including a phosphatidic acid phosphatase (PAP), dephosphorylating phosphatidic acid (PA) into diacylglycerol (DAG), and MGD1, transferring a galactose onto DAG thus generating monogalactosyldiacylglycerol (MGDG). It has been shown that PA and DAG could be synthesized in the plastid via the so-called 'prokaryotic' pathway or imported from the endoplasmic reticulum via the 'eukaryotic' pathway. In vitro studies support the existence of (1) a negative regulation of the plastid PAP by DAG and (2) an activation of MGD1 by PA. We developed a mathematical model of the IEM galactolipid biosynthesis pathway to understand the properties of the system ruled by the presence of these two regulatory motifs. We demonstrated that the design of the system implies that PA should accumulate to levels that are not observed experimentally, regardless of its prokaryotic or eukaryotic origin. PA should therefore be used for other syntheses, such as that of phosphatidylglycerol. Whereas a massive influx of eukaryotic PA appears unlikely, an influx of eukaryotic DAG in the IEM is supported by simulations. The model also implies that DAG cannot transiently accumulate and that PA mainly acts as a signal switching the whole system on. Eventually, this analysis highlights the fact that the PAP enzyme could easily become dispensable and that the design of the system, with the two regulatory motifs, could precede the loss of the PAP gene or activity in this pathway, a phenomenon that occurred independently in most clades of Angiosperms.
[Show abstract][Hide abstract] ABSTRACT: INTRODUCTION
Chlamydomonas reinhardtii is extensively used as a model organism for exploring fundamental processes in photosynthesis and lipid transesterification. Laser ablation electrospray ionization (LAESI) mass spectrometry (MS) has been applied for the in situ metabolic and lipid profiling of microalgae under varied environmental conditions. Direct analyses of live microalgae enable the evaluation of metabolic network models, and advance our understanding of cellular responses and cell differentiation in relation to environmental factors. Stable isotope pulse-chase analysis by MS allows for time course profiling of labeled metabolites and the determination of molecular turnover rates under varied stress conditions. In this study, pulse-chase experiments were monitored by LAESI-MS to determine the metabolic and lipid turnover rates in small cell populations of C. reinhardtii.
For the pulse phase, wild type C. reinhardtii was cultured in 15N-labeled tris-acetate phosphate (TAP) medium and incubated at 27°C in a shaker running at 80 rpm for 96 hours. The chase phase was initiated by replacing the 15N-labeled TAP with and excess of unlabeled TAP containing 14N compounds. During the chase phase, sampling was performed at various time points followed by LAESI-MS analysis. Mass spectra were acquired by a quadrupole time-of- flight system. For evaluating the abundances of labeled compounds, the overlapping natural isotope peak intensities were manually subtracted. Following the time course of exchanging the 15N-labeled metabolites and lipids in the sample by their 14N counterparts provided insight into the turnover rates of these molecules.
We first evaluated the effect of nitrogen isotopic composition on the growth and development of C. reinhardtii. By comparison with the 14N-TAP control, our observations indicated that C.
reinhardtii grew in the 15N-TAP medium without any apparent physiological stress responses. No morphological differences between cells grown in 15N-TAP and 14N-TAP media were observed and they showed similar growth rates.
To explore the turnover rates of the abundant diacylglyceryl-N,N,N-trimethylhomoserines (DGTS) in C. reinhardtii, we focused on the corresponding peaks present between m/z 700 and 750. These lipids contained a single nitrogen atom, and the mass spectra in the pulse phase demonstrated that the C. reinhardtii cells successfully incorporated 15N into these molecules from the 15N-TAP media. Identification of the acyl chain lengths and verification of the 15N incorporation was achieved by tandem MS. For example, the ion assigned as DGTS(16:0/18:3) had a m/z 735 molecular ion and related lyso-lipid fragments at m/z 497 and 475 in 15N-TAP media, and a m/z 734 molecular ion and related lyso-lipid fragments of m/z 496 and 474 in 14N- TAP media.
Peak area ratios for m/z 735 and 734 as a function of time showed a decay that followed first order kinetics. Half-lives for the conversion of 15N-labeled DGTS(16:0/18:3) and DGTS(34:4) back to their unlabeled forms were 4.35 h and 5.70 h, respectively. The corresponding rate coefficients were 6.4×10-5 s-1 and 4.9×10-5 s-1. We observed similar kinetics for other nitrogen containing compounds and their rate coefficients were determined. LAESI-MS in combination with stable isotope pulse-chase experiments promises to provide a rapid assessment of metabolic conversion rates in complex biological systems.
62nd ASMS National Conference, Baltimore, MD; 07/2014
[Show abstract][Hide abstract] ABSTRACT: The microalga Chlamydomonas reinhardtii has been extensively studied for its relevance in biofuel production. This fully sequenced model organism has applications in focused areas such as photosynthesis, metabolism, cell cycle, motility, and genetics.1 To improve the understanding of lipid biosynthesis and its implications for using Chlamydomonas as a biofuel source, factors such as environmental conditions and selection of strains with particular genotypes need to be further investigated. Depending on nutrient availability and environmental conditions C. reinhardtii has the ability to store energy as starch or as neutral lipids, e.g., triacylglycerols (TAG). Here we demonstrate the use of a robust ambient analytical tool, laser ablation electrospray ionization (LAESI) mass spectrometry (MS) with ion mobility separation (IMS), to better understand metabolic changes and adaptations at the biochemical pathway level under varied stress conditions.
C. reinhardtii mutants with an impaired starch pathway were investigated for the production of neutral storage lipids under altered light conditions. Starch synthesis is inhibited by disrupting the central enzyme of ADP-glucose pyrophosphorylase (AGPase-SS) that is responsible for the formation of glucosyl nucleotides from glucose-1-phosphate with ATP in the starch pathway. This enzyme is activated by 3-phosphoglyceric acid (3-PGA). The Sta1 mutant has a disrupted large catalytic subunit of AGPase-SS, and as a consequence it exhibits a reduced activation by 3-PGA. This allows the mutant to retain less than ~10% of its normal starch production.2 The Sta6 mutant has a disrupted small catalytic subunit of AGPase-SS and as a result only retains ~1% of the normal starch levels.3
Conventional lipid analysis of microalgae consists of extensive extraction protocols and/or derivatization, resulting in lengthy runs and the loss of some structural information. To reduce the complexity of the sample, a separation step that distinguishes the different classes of compounds and isobaric species is required. LAESI-MS is an ambient ionization technique that has been employed to detect metabolites, lipids, and peptides from diverse biological samples. This technique utilizes the natural water content of cells and tissues to facilitate laser energy deposition by the strong absorption of water at 2.94 μm wavelength and to produce an ablation plume. The plume is then ionized by an electrospray and sampled by a high-performance Q- TOF mass spectrometer (Synapt G2 S, Waters Co.). A typical LAESI mass spectrum of Chlamydomonas contains approximately ~250 detected ions but it does not differentiate between structural isomers and isobaric species. Combination of LAESI-MS with IMS has been shown to produce a 3 fold increase in molecular coverage.4 The resulting enhanced data includes m/z ratios, drift times (DT) related to the collision cross section of the ion, and peak intensities. To reduce spectral interferences from the medium and to prevent osmotic shock the cells are deposited in a silica membrane spin tube and the supernatant is separated from the cells by centrifuging at 2,000×g for 1 min. The laser pulses are directly coupled into the remaining pellet of microalgae on the filter.
Initially the wild type (WT) C. reinhardtii was studied under low and high light conditions, with photosynthetically active radiation (PAR) 0 and 150 μmolm-2sec-1, respectively. After a 72 h period, the cells were harvested and LAESI-IMS-MS was performed. In the lipid region of the spectra, normalized for the most abundant diacylglyceryl-N,N,N-trimethylhomoserine (DGTS) ion, DGTS(34:3), enhanced production of monogalactosyldiacylglycerols (MGDG), digalactosyldiacylglycerols (DGDG) and TAG lipids was observed under high light conditions.
The Sta1 and Sta6 mutants were investigated for lipid production in altered light conditions ranging from 0 to 200 μmolm-2sec-1 over a 72 h time period. When compared to the WT cells, significant levels of TAG and DGDG were observed in both mutants throughout the 72 h time period. Using LAESI-MS without IMS ~10 TAG lipid species were detected. The introduction of IMS improved the molecular coverage of TAG lipids to ~50 species in the DT range of 189 to 204 ms (see Figure 1a).
When compared to WT, the Sta1 mutant showed stronger ion signal for TAG lipids within 800 < m/z < 880 at 72 h under high light condition. Conversely, the Sta6 mutant presented the strongest ion signal for TAG lipids at 72 h in low light illumination. Under opposite lighting conditions, the two Sta mutants exhibited similar TAG lipid profiles.
To reduce spectral interferences and differentiate close to isobaric species, for example TAG(54:7) and the 13C peak of chlorophyll a, both at nominal m/z 894, two DT ranges were inspected. In the LAESI-IMS-MS spectrum, the DT range integrated between 201 and 207 ms revealed the TAG(54:7) species at m/z 894.7571, whereas the DT range integrated between 181 and 188 ms showed the 13C isotope peak of chlorophyll a at m/z 894.5512 (see Figure 1b).
These results demonstrate our ability to observe numerous changes in lipid levels in WT and genetically modified microalgae populations affected by light exposure. We found that impaired starch pathways C. reinhardtii result in energy storage redirected to neutral lipids. These capabilities are expected to accelerate the research on the utility of genetically altered microalgae strains for biofuel production.
the 62nd ASMS National Conference, Baltimore, MD; 07/2014
[Show abstract][Hide abstract] ABSTRACT: The understanding of chloroplast function requires the precise localization of proteins in each of its sub-compartments. High-sensitivity mass spectrometry has allowed the inventory of proteins in thylakoid, stroma, and envelope fractions. Concerning membrane association, proteins can be either integral or peripheral or even soluble proteins bound transiently to a membrane complex. We sought a method providing information at the surface of the outer envelope membrane (OEM), based on specific tagging with biotin or proteolysis using thermolysin, a non-membrane permeable protease. To evaluate this method, envelope, thylakoid, and stroma proteins were separated by two-dimensional electrophoresis and analyzed by immunostaining and mass spectrometry. A short selection of proteins associated to the chloroplast envelope fraction was checked after superficial treatments of intact chloroplasts. We showed that this method could allow the characterization of OEM embedded proteins facing the cytosol, as well as peripheral and soluble proteins associated via tight or lose interactions. Some stromal proteins were associated with biotinylated spots and analyzes are still needed to determine whether polypeptides were tagged prior import or if they co-migrated with OEM proteins. This method also suggests that some proteins associated with the inner envelope membrane (IEM) might need the integrity of a trans-envelope (IEM-OEM) protein complex (e.g., division ring-forming components) or at least an intact OEM partner. Following this evaluation, proteomic analyzes should be refined and the putative role of inter-membrane space components stabilizing trans-envelope complexes demonstrated. For future comprehensive studies, perspectives include the dynamic analyses of OEM proteins and IEM-OEM complexes in various physiological contexts and using virtually any other purified membrane organelle.
[Show abstract][Hide abstract] ABSTRACT: Probing molecules using perdeuteration (i.e deuteration in which all hydrogen atoms are replaced by deuterium) is extremely useful in a wide range of biophysical techniques. In the case of lipids, the synthesis of the biologically relevant unsaturated perdeuterated lipids is challenging and not usually pursued. In this work, perdeuterated phospholipids and sterols from the yeast Pichia pastoris grown in deuterated medium are extracted and analyzed as derivatives by gas chromatography and mass spectrometry respectively. When yeast cells are grown in a deuterated environment, the phospholipid homeostasis is maintained but the fatty acid unsaturation level is modified while the ergosterol synthesis is not affected by the deuterated culture medium. Our results confirm that the production of well defined natural unsaturated perdeuterated lipids is possible and gives also new insights about the process of desaturase enzymes.
PLoS ONE 04/2014; 9(4):e92999. DOI:10.1371/journal.pone.0092999 · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Thylakoid membranes, the universal structure where photosynthesis takes place in all oxygenic photosynthetic organisms from cyanobacteria to higher plants, have a unique lipid composition. They contain a high fraction of 2 uncharged glycolipids, the galactoglycerolipids mono- and digalactosyldiacylglycerol (MGDG and DGDG, respectively), and an anionic sulfolipid, sulfoquinovosediacylglycerol (SQDG). A remarkable feature of the evolution from cyanobacteria to higher plants is the conservation of MGDG, DGDG, SQDG, and phosphatidylglycerol (PG), the major phospholipid of thylakoids. Using neutron diffraction on reconstituted thylakoid lipid extracts, we observed that the thylakoid lipid mixture self-organizes as a regular stack of bilayers. This natural lipid mixture was shown to switch from hexagonal II toward lamellar phase on hydration. This transition and the observed phase coexistence are modulated by the fine-tuning of the lipid profile, in particular the MGDG/DGDG ratio, and by the hydration. Our analysis highlights the critical role of DGDG as a contributing component to the membrane stacking via hydrogen bonds between polar heads of adjacent bilayers. DGDG interactions balance the repulsive electrostatic contribution of the charged lipids PG and SQDG and allow the persistence of regularly stacked membranes at high hydration. In developmental contexts or in response to environmental variations, these properties can contribute to the highly dynamic flexibility of plastid structure.-Demé, B., Cataye, C., Block, M. A., Maréchal, E., Jouhet, J. Contribution of galactoglycerolipids to the 3-dimensional architecture of thylakoids.
The FASEB Journal 04/2014; 28(8). DOI:10.1096/fj.13-247395 · 5.48 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Mono- and digalactosyldiacylglycerol (MGDG and DGDG) are the most abundant lipids of photosynthetic membranes (thylakoids). In Arabidopsis green tissues, MGD1 is the main enzyme synthesizing MGDG. This monotopic enzyme is embedded in the inner envelope membrane of chloroplasts. DGDG synthesis occurs in the outer envelope membrane. Although the suborganellar localization of MGD1 has been determined, it is still not known how the lipid/glycolipid composition influences its binding to the membrane. The existence of a topological relationship between MGD1 and "embryonic" thylakoids is also unknown. To investigate MGD1 membrane binding, we used a Langmuir membrane model allowing the tuning of both lipid composition and packing. Surprisingly, MGD1 presents a high affinity to MGDG, its product, which maintains the enzyme bound to the membrane. This positive feedback is consistent with the low level of diacylglycerol, the substrate of MGD1, in chloroplast membranes. By contrast, MGD1 is excluded from membranes highly enriched in, or made of, pure DGDG. DGDG therefore exerts a retrocontrol, which is effective on the overall synthesis of galactolipids. Previously identified activators, phosphatidic acid and phosphatidylglycerol, also play a role on MGD1 membrane binding via electrostatic interactions, compensating the exclusion triggered by DGDG. The opposite effects of MGDG and DGDG suggest a role of these lipids on the localization of MGD1 in specific domains. Consistently, MGDG induces the self-organization of MGD1 into elongated and reticulated nanostructures scaffolding the chloroplast membrane.-Sarkis, J., Rocha, J., Maniti, O., Jouhet, J., Vié, V., Block, M. A., Breton, C., Maréchal, E., Girard-Egrot, A. The influence of lipids on MGD1 membrane binding highlights novel mechanisms for galactolipid biosynthesis regulation in chloroplasts.
The FASEB Journal 04/2014; 28(7). DOI:10.1096/fj.14-250415 · 5.48 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Glycerolipids constituting the matrix of photosynthetic membranes, from cyanobacteria to chloroplasts of eukaryotic cells, comprise monogalactosyldiacylglycerol, digalactosyldiacylglycerol, sulfoquinovosyldiacylglycerol and phosphatidylglycerol. This review covers our current knowledge on the structural and functional features of these lipids in various cellular models, from prokaryotes to eukaryotes. Their relative proportions in thylakoid membranes result from highly regulated and compartmentalized metabolic pathways, with a cooperation, in the case of eukaryotes, of non plastidic compartments. This review also focuses on the role of each of these thylakoid glycerolipids in stabilizing protein complexes of the photosynthetic machinery, which might be one of the reasons for their fascinating conservation in the course of evolution. This article is part of a Special Issue entitled: Dynamic and ultrastructure of bioenergetic membranes and their components.
[Show abstract][Hide abstract] ABSTRACT: Photosynthetic membranes have a unique lipid composition that has been remarkably well conserved from cyanobacteria to chloroplasts. These membranes are characterized by a very high content in galactoglycerolipids, i.e., mono- and digalactosyldiacylglycerol (MGDG and DGDG, respectively). Galactoglycerolipids make up the bulk of the lipid matrix in which photosynthetic complexes are embedded. They are also known to fulfill specific functions, such as stabilizing photosystems, being a source of polyunsaturated fatty acids for various purposes and, in some eukaryotes, being exported to other subcellular compartments. The conservation of MGDG and DGDG suggests that selection pressures might have conserved the enzymes involved in their biosynthesis, but this does not appear to be the case. Important evolutionary transitions comprise primary endosymbiosis (from a symbiotic cyanobacterium to a primary chloroplast) and secondary endosymbiosis (from a symbiotic unicellular algal eukaryote to a secondary plastid). In this review, we compare biosynthetic pathways based on available molecular and biochemical data, highlighting enzymatic reactions that have been conserved and others that have diverged or been lost, as well as the emergence of parallel and alternative biosynthetic systems originating from other metabolic pathways. Questions for future research are highlighted.
Progress in lipid research 03/2014; DOI:10.1016/j.plipres.2014.02.001 · 12.96 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A piperidinyl-benzimidazolidinone scaffold has been found in the structure of different inhibitors of membrane glycerolipid metabolism, acting on enzymes manipulating diacylglycerol or phosphatidic acid. Screening a focus library of piperidinyl-benzimidazolidinone analogs might therefore identify compounds acting against infectious parasites. We first evaluated the in vitro effects of (S)-2-(dibenzylamino)-3-phenylpropyl 4-(1,2-dihydro-2-oxobenzo[d]imidazol-3-yl)piperidine-1-carboxylate (compound 1) on Toxoplasma gondii and Plasmodium falciparum. In T. gondii, motility and apical complex integrity appeared unaffected, whereas cell division was inhibited at concentrations of compound 1 in the micromolar range. In P. falciparum, proliferation of erythrocytic stages was inhibited, without any delayed death phenotype. We then explored a library of 250 analogs in two steps. We selected 114 compounds with an IC50 cutoff of 2 μM on at least one species and determined in vitro selectivity indexes (SI), based on toxicity against K-562 human cells. We identified compounds with high gains in IC50 (in the 100 nM range) and SI (up to 1,000-2,000). Isobole analyses of two of the most active compounds against P. falciparum indicated that their interaction with artemisin was additive. We propose structure activity relationship (SAR) models, which will be useful for the design of probes to identify the compounds' target(s), and optimizations for mono- or combined-therapeutic strategies.
[Show abstract][Hide abstract] ABSTRACT: The knowledge of the membrane lipid metabolism in photosynthetic cells is expected to benefit from the availability of inhibitors acting at the level of specific enzymes like MGD1 (E.C. 184.108.40.206) that catalyzes the synthesis of monogalactosyldiacylglycerol (MGDG) in chloroplasts. MGDG is a major lipid of photosynthetic membrane, interacting with photosystems. It is the precursor of digalactosyldiacylglycerol that serves as a phospholipid surrogate when plants are deprived of phosphate, and it is a source of polyunsaturated fatty acids for jasmonic acid syntheses. MGD1 is activated by phosphatidic acid and thus a coupling point between phospholipid and galactolipid metabolisms. Here we describe a method to screen for inhibitors of MGD1 assayed in liposomes. Selected compounds can therefore reach the core of the biological membranes in which the target sits. We then describe a secondary screen to evaluate the efficiency of developed compounds at the whole plant level. Major issues raised by the screening of inhibitors acting on membrane proteins are discussed and can be useful for similar targets.