Stepwise Biogenesis of Subpopulations of Lipid Droplets in Nitrogen Starved Phaeodactylum tricornutum Cells.

Diatoms are unicellular heterokonts, living in oceans and freshwaters, exposed to frequent environmental variations. They have a sophisticated membrane compartmentalization and are bounded by a siliceous cell-wall. Formation of lipid droplets (LDs), filled with triacylglycerol (TAG), is a common response to stress. The proteome of mature-LDs from Phaeodactylum tricornutum highlighted the lack of proteins involved in early-LD formation, TAG biosynthesis or LD-to-LD connections. These features suggest that cytosolic LDs might reach a size limit. We analyzed the dynamics of LD formation in P. tricornutum (Pt1 8.6; CCAP 1055/1) during 7 days of nitrogen starvation, by monitoring TAG by mass spectrometry-based lipidomics, and LD radius using epifluorescence microscopy and pulse field gradient nuclear magnetic resonance. We confirmed that mature LDs reach a maximal size. Based on pulse field gradient nuclear magnetic resonance, we did not detect any LD-LD fusion. Three LD subpopulations were produced, each with a different maximal size, larger-sized LDs (radius 0.675 ± 0.125 µm) being generated first. Mathematical modeling showed how smaller LDs are produced once larger LDs have reached their maximum radius. In a mutant line having larger cells, the maximal size of the first LD subpopulation was higher (0.941 ± 0.169 µm), while the principle of stepwise formation of distinct LD populations was maintained. Results suggest that LD size is determined by available cytosolic space and sensing of an optimal size reached in the previous LD subpopulation. Future perspectives include the unraveling of LD-size control mechanisms upon nitrogen shortage. This study also provides novel prospects for the optimization of oleaginous microalgae for biotechnological applications.

Growth Mechanism and Surface State of CuInS2 Nanocrystals Synthesized with Dodecanethiol.

Ternary metal chalcogenide nanocrystals (NCs) offer exciting opportunities as novel materials to be explored on the nanoscale showing optoelectronic properties tunable with size and composition. CuInS2 (CIS) NCs are the most widely studied representatives of this family as they can be easily prepared with good size control and in high yield by reacting the metal precursors (copper iodide and indium acetate) in dodecanethiol (DDT). Despite the widespread use of this synthesis method, both the reaction mechanism and the surface state of the obtained NCs remain elusive. Here, we perform in situ X-ray diffraction using synchrotron radiation to monitor the pre- and postnucleation stages of the formation of CIS NCs. SAXS measurements show that the reaction intermediate formed at 100 °C presents a periodic lamellar structure with a characteristic spacing of 34.9 Å. WAXS measurements performed after nucleation of the CIS NCs at 230 °C demonstrate that their growth kinetics depend on the degree of precursor conversion achieved in the initial stage at 100 °C. NC formation requires the cleavage of S-C bonds. We reveal by means of combined 1D and 2D proton and carbon NMR analyses that the generated dodecyl radicals lead to the formation of a new thioether species R-S-R. The latter is part of a ligand double layer, which consists of dynamically bound dodecanethiolate ligands as well as of head-to-tail bound R-S-R molecules. This ligand double layer and a high ligand density (3.6 DDT molecules per nm2) are at the origin of the apparent difficulty to functionalize the surface of CIS NCs obtained with the DDT method.

The structural organization of seed oil bodies could explain the contrasted oil extractability observed in two rapeseed genotypes.

MAIN CONCLUSION: The protein, phospholipid and sterol composition of the oil body surface from the seeds of two rapeseed genotypes was compared in order to explain their contrasted oil extractability. In the mature seeds of oleaginous plants, storage lipids accumulate in specialized structures called oil bodies (OBs). These organelles consist of a core of neutral lipids surrounded by a phospholipid monolayer in which structural proteins are embedded. The physical stability of OBs is a consequence of the interactions between proteins and phospholipids. A detailed study of OB characteristics in mature seeds as well as throughout seed development was carried out on two contrasting rapeseed genotypes Amber and Warzanwski. These two accessions were chosen because they differ dramatically in (1) crushing ability, (2) oil extraction yield and, (3) the stability of purified OBs. Warzanwski has higher crushing ability, better oil extraction yield and less stable purified OBs than Amber. OB morphology was investigated in situ using fluorescence microscopy, transmission electron microscopy and pulsed field gradient NMR. During seed development, OB diameter first increased and then decreased 30 days after pollination in both Amber and Warzanwski embryos. In mature seeds, Amber OBs were significantly smaller. The protein, phospholipid and sterol composition of the hemi-membrane was compared between the two accessions. Amber OBs were enriched with H-oleosins and steroleosins, suggesting increased coverage of the OB surface consistent with their higher stability. The nature and composition of phospholipids and sterols in Amber OBs suggest that the hemi-membrane would have a more rigid structure than that of Warzanwski OBs.

In vivo measurement of the size of oil bodies in plant seeds using a simple and robust pulsed field gradient NMR method.

An easy to implement and convenient method to measure the mean size of oil bodies (OBs) in plant seeds is proposed using a pulsed field gradient nuclear magnetic resonance (PFGNMR) approach. PFGNMR is a well-known technique used to study either free or restricted diffusion of molecules. As triacylglycerols (TAG) are confined in OBs, analysis of their diffusion properties is a well-suited experimental approach to determine OB sizes. In fact, at long diffusion time, TAG mean squared displacement is limited by the size of the domain where these molecules are confined. In order to access the OB size distribution, strong intensities of magnetic field gradients are generally required. In this work we demonstrate for the first time that a standard liquid-phase NMR probe equipped with a weak-intensity gradient coil can be used to determine the mean size of OBs. Average sizes were measured for several seeds, and OB diameters obtained by PFGNMR were fully consistent with previously published values obtained by microscopy techniques. Moreover, this approach provided evidence of TAG transfer through the network of interconnected OBs, which is dependent on the ability of adjacent membranes to open diffusion routes between OBs. The main advantage of the NMR method is that it does not require any sample preparation and experiments are performed with whole seeds directly introduced in a standard NMR tube.

Enhanced charge separation in ternary P3HT/PCBM/CuInS2 nanocrystals hybrid solar cells.

Geminate recombination of bound polaron pairs at the donor/acceptor interface is one of the major loss mechanisms in organic bulk heterojunction solar cells. One way to overcome Coulomb attraction between opposite charge carriers and to achieve their full dissociation is the introduction of high dielectric permittivity materials such as nanoparticles of narrow band gap semiconductors. We selected CuInS2 nanocrystals of 7.4 nm size, which present intermediate energy levels with respect to poly(3-hexylthiophene) (P3HT) and Phenyl-C61-butyric acid methyl ester (PCBM). Efficient charge transfer from P3HT to nanocrystals takes place as evidenced by light-induced electron spin resonance. Charge transfer between nanocrystals and PCBM only occurs after replacing bulky dodecanethiol (DDT) surface ligands with shorter 1,2-ethylhexanethiol (EHT) ligands. Solar cells containing in the active layer a ternary blend of P3HT:PCBM:CuInS2-EHT nanocrystals in 1:1:0.5 mass ratio show strongly improved short circuit current density and a higher fill factor with respect to the P3HT:PCBM reference device. Complementary measurements of the absorption properties, external quantum efficiency and charge carrier mobility indicate that enhanced charge separation in the ternary blend is at the origin of the observed behavior. The same trend is observed for blends using the glassy polymer poly(triarylamine) (PTAA).

Quenching dynamics in CdSe nanoparticles: surface-induced defects upon dilution.

We have analyzed the decays of the fluorescence of colloidal CdSe quantum dots (QDs) suspensions during dilution and titration by the ligands. A ligand shell made of a combination of trioctylphosphine (TOP), oleylamine (OA), and stearic acid (SA) stabilizes the as-synthesized QDs. The composition of the shell was analyzed and quantified using high resolution liquid state 1H nuclear magnetic resonance (NMR) spectroscopy. A quenching of the fluorescence of the QDs is observed upon removal of the ligands by diluting the stock solution of the QDs. The fluorescence is restored by the addition of TOP. We analyze the results by assuming a binomial distribution of quenchers among the QDs and predict a linear trend in the time-resolved fluorescence decays. We have used a nonparametric analysis to show that for our QDs, 3.0 ± 0.1 quenching sites per QD on average are revealed by the removal of TOP. We moreover show that the quenching rates of the quenching sites add up. The decay per quenching site can be compared with the decay at saturation of the dilution effect. This provides a value of 2.88 ± 0.02 for the number of quenchers per QD. We extract the quenching dynamics of one site. It appears to be a process with a distribution of rates that does not involve the ligands.

Massive production of butanediol during plant infection by phytopathogenic bacteria of the genera Dickeya and Pectobacterium.

Plant pathogenic bacteria of the genera Dickeya and Pectobacterium are broad-host-range necrotrophs which cause soft-rot diseases in important crops. A metabolomic analysis, based on (13)C-NMR spectroscopy, was used to characterize the plant-bacteria interaction. Metabolic profiles revealed a decline in plant sugars and amino acids during infection and the concomitant appearance of a compound identified as 2,3-butanediol. Butanediol is the major metabolite found in macerated tissues of various host plants. It is accumulated during the symptomatic phase of the disease. Different species of Dickeya or Pectobacterium secrete high levels of butanediol during plant infection. Butanediol has been described as a signalling molecule involved in plant/bacterium interactions and, notably, able to induce plant systemic resistance. The bud genes, involved in butanediol production, are conserved in the phytopathogenic enterobacteria of the genera Dickeya, Pectobacterium, Erwinia, Pantoea and Brenneria. Inactivation of the bud genes of Dickeya dadantii revealed that the virulence of budA, budB and budR mutants was clearly reduced. The genes budA, budB and budC are highly expressed during plant infection. These data highlight the importance of butanediol metabolism in limiting acidification of the plant tissue during the development of the soft-rot disease caused by pectinolytic enterobacteria.

Toward Arabidopsis thaliana hydrophilic metabolome: assessment of extraction methods and quantitative 1H NMR.

Our goal was to establish the hydrophilic metabolome of heterotrophic Arabidopsis thaliana cells grown in suspension, a cellular model of plant sink tissues. Water-soluble metabolites were extracted using four protocols: perchloric acid, boiling ethanol, methanol and methanol/chloroform (M/Chl). They were detected and quantified using (1)H nuclear magnetic resonance (NMR) spectroscopy at 400 MHz. Extraction yields and reproducibility of the extraction methods were investigated. The effects of cell harvest protocol, cell grinding and lyophilization and storage conditions on the measured metabolic profiles were also studied. These quantitative studies demonstrated for the first time that the four extraction protocols commonly used do lead to quite similar molecular compositions as analyzed by (1)H NMR. The M/Chl method proved effective and reliable to prepare series of physiologically significant extracts from plant cells for (1)H NMR analysis. Reproducibility of the detected metabolome was assessed over long periods of time by analyzing a large number of separate extracts prepared from independent cultures. Larger variations in the NMR metabolite profiles could be correlated to changes in physiological parameters of the culture medium. Quantitative resolved (1)H NMR of cell extracts proved to be robust and reliable for routine metabolite profiling of plant cell cultures.

In plants, 3-o-methylglucose is phosphorylated by hexokinase but not perceived as a sugar.

In plants, sugars are the main respiratory substrates and important signaling molecules in the regulation of carbon metabolism. Sugar signaling studies suggested that sugar sensing involves several key components, among them hexokinase (HXK). Although the sensing mechanism of HXK is unknown, several experiments support the hypothesis that hexose phosphorylation is a determining factor. Glucose (Glc) analogs transported into cells but not phosphorylated are frequently used to test this hypothesis, among them 3-O-methyl-Glc (3-OMG). The aim of the present work was to investigate the effects and fate of 3-OMG in heterotrophic plant cells. Measurements of respiration rates, protein and metabolite contents, and protease activities and amounts showed that 3-OMG is not a respiratory substrate and does not contribute to biosynthesis. Proteolysis and lipolysis are induced in 3-OMG-fed maize (Zea mays L. cv DEA) roots in the same way as in sugar-starved organs. However, contrary to the generally accepted idea, phosphorous and carbon nuclear magnetic resonance experiments and enzymatic assays prove that 3-OMG is phosphorylated to 3-OMG-6-phosphate, which accumulates in the cells. Insofar as plant HXK is involved in sugar sensing, these findings are discussed on the basis of the kinetic properties because the catalytic efficiency of HXK isolated from maize root tips is five orders of magnitude lower for 3-OMG than for Glc and Man.

Sucrose cycling in heterotrophic plant cell metabolism: first step towards an experimental model.

Sucrose is the cornerstone of higher plant metabolism. Produced by photosynthesis, sucrose is the main substrate for respiration and biosynthesis. The emerging idea is that sucrose may act as regulator of its own metabolism, characterized in particular by a permanent process of degradation and formation. This sucrose turnover may control several important physiological functions. Of particular concern is an energy dependent cycle involving the hexokinase. This report presents an experimental approach to define quantitatively physiological states of suspension-cultured plant cells wih reference to their sucrose content and respiration rate. Sucrose depletion of normal cells incubated in a medium devoid of sugar is measured in vivo using 13C and respiration is simultaneously recorded. Results obtained with sucrose-storing cells and Arabidopsis thaliana show that respiration rate is closely linked to the available sucrose. Sucrose-depleted cells offer a stable model to study the bioenergetics of the process.