Mitochondrial metabolism in developing embryos of Brassica napus

The metabolism of developing plant seeds is directed toward transforming primary assimilatory products (sugars and amino acids) into seed storage compounds. To understand the role of mitochondria in this metabolism, metabolic fluxes were determined in developing embryos of Brassica napus. After labeling with [1,2-(13)C2]glucose + [U-(13)C6]glucose, [U-(13)C3]alanine, [U-(13)C5]glutamine, [(15)N]alanine, (amino)-[(15)N]glutamine, or (amide)-[(15)N]glutamine, the resulting labeling patterns in protein amino acids and in fatty acids were analyzed by gas chromatography-mass spectrometry. Fluxes through mitochondrial metabolism were quantified using a steady state flux model. Labeling information from experiments using different labeled substrates was essential for model validation and reliable flux estimation. The resulting flux map shows that mitochondrial metabolism in these developing seeds is very different from that in either heterotrophic or autotrophic plant tissues or in most other organisms: (i) flux around the tricarboxylic acid cycle is absent and the small fluxes through oxidative reactions in the mitochondrion can generate (via oxidative phosphorylation) at most 22% of the ATP needed for biosynthesis; (ii) isocitrate dehydrogenase is reversible in vivo; (iii) about 40% of mitochondrial pyruvate is produced by malic enzyme rather than being imported from the cytosol; (iv) mitochondrial flux is largely devoted to providing precursors for cytosolic fatty acid elongation; and (v) the uptake of amino acids rather than anaplerosis via PEP carboxylase determines carbon flow into storage proteins.     

Cellular and clinical report of new Griscelli syndrome type III cases

The RAB27A/Melanophilin/Myosin-5a tripartite protein complex is required for capturing mature melanosomes in the peripheral actin network of melanocytes for subsequent transfer to keratinocytes. Mutations in any one member of this tripartite complex cause three forms of Griscelli syndrome (GS), each with distinct clinical features but with a similar cellular phenotype. To date, only one case of GS type III (GSIII), caused by mutations in the Melanophilin (MLPH) gene, has been reported. Here, we report seven new cases of GSIII in three distinct Arab pedigrees. All affected individuals carried a homozygous missense mutation (c.102C>T; p.R35W), located in the conserved Slp homology domain of MLPH, and had hypomelanosis of the skin and hair. We report the first cellular studies on GSIII melanocytes, which demonstrated that MLPH(R35W) causes perinuclear aggregation of melanosomes in melanocytes, typical for GS. Additionally, co-immunoprecipitation assays showed that MLPH(R35W) lost its interaction with RAB27A, indicating pathogenicity of the R35W mutation.     

Sperm epidermal growth factor receptor (EGFR) mediates α7 acetylcholine receptor (AChR) activation to promote fertilization

To attain fertilization the spermatozoon binds to the egg zona pellucida (ZP) via sperm receptor(s) and undergoes an acrosome reaction (AR). Several sperm receptors have been described in the literature; however, the identity of this receptor is not yet certain. In this study, we suggest that the α7 nicotinic acetylcholine receptor (α7nAChR) might be a sperm receptor activated by ZP to induce epidermal growth factor receptor (EGFR)-mediated AR. We found that isolated ZP or α7 agonists induced the AR in sperm from WT but not α7-null spermatozoa, and the induced AR was inhibited by α7 or EGFR antagonists. Moreover, α7-null sperm showed very little binding to the egg, and microfluidic affinity in vitro assay clearly showed that α7nAChR, as well as EGFR, interacted with ZP3. Induction of EGFR activation and the AR by an α7 agonist was inhibited by a Src family kinase (SFK) inhibitor. In conclusion we suggest that activation of α7 by ZP leads to SFK-dependent EGFR activation, Ca(2+) influx, and the acrosome reaction.     

A “Shallow Phylogeny” of Shallow Barnacles (Chthamalus)

Background

We present a multi-locus phylogenetic analysis of the shallow water (high intertidal) barnacle genus Chthamalus, focusing on member species in the western hemisphere. Understanding the phylogeny of this group improves interpretation of classical ecological work on competition, distributional changes associated with climate change, and the morphological evolution of complex cirripede phenotypes.

Methodology and Findings

We use traditional and Bayesian phylogenetic and ‘deep coalescent’ approaches to identify a phylogeny that supports the monophyly of the mostly American ‘fissus group’ of Chthamalus, but that also supports a need for taxonomic revision of Chthamalus and Microeuraphia. Two deep phylogeographic breaks were also found within the range of two tropical American taxa (C. angustitergum and C. southwardorum) as well.

Conclusions

Our data, which include two novel gene regions for phylogenetic analysis of cirripedes, suggest that much more evaluation of the morphological evolutionary history and taxonomy of Chthamalid barnacles is necessary. These data and associated analyses also indicate that the radiation of species in the late Pliocene and Pleistocene was very rapid, and may provide new insights toward speciation via transient allopatry or ecological barriers.     

The role of light in soybean seed filling metabolism

Soybean (Glycine max) yields high levels of both protein and oil, making it one of the most versatile and important crops in the world. Light has been implicated in the physiology of developing green seeds including soybeans but its roles are not quantitatively understood. We have determined the light levels reaching growing soybean embryos under field conditions and report detailed redox and energy balance analyses for them. Direct flux measurements and labeling patterns for multiple labeling experiments including [U‐¹³C₆]‐glucose, [U‐¹³C₅]‐glutamine, the combination of [U‐¹⁴C₁₂]‐sucrose + [U‐¹⁴C₆]‐glucose + [U‐¹⁴C₅]‐glutamine + [U‐¹⁴C₄]‐asparagine, or ¹⁴CO₂ labeling were performed at different light levels to give further insight into green embryo metabolism during seed filling and to develop and validate a flux map. Labeling patterns (protein amino acids, triacylglycerol fatty acids, starch, cell wall, protein glycan monomers, organic acids), uptake fluxes (glutamine, asparagine, sucrose, glucose), fluxes to biomass (protein amino acids, oil), and respiratory fluxes (CO₂, O₂) were established by a combination of gas chromatography‐mass spectrometry, ¹³C‐ and ¹H‐NMR, scintillation counting, HPLC, gas chromatography‐flame ionization detection, C:N and amino acid analyses, and infrared gas analysis, yielding over 750 measurements of metabolism. Our results show: (i) that developing soybeans receive low but significant light levels that influence growth and metabolism; (ii) a role for light in generating ATP but not net reductant during seed filling; (iii) that flux through Rubisco contributes to carbon conversion efficiency through generation of 3‐phosphoglycerate; and (iv) a larger contribution of amino acid carbon to fatty acid synthesis than in other oilseeds analyzed to date.     

Identification and Characterization of gshA,a Gene Encoding the Glutamate-Cysteine Ligase in the Halophilic Archaeon Haloferax volcanii

Halophilic archaea were found to contain in their cytoplasm millimolar concentrations of γ-glutamylcysteine (γGC) instead of glutathione. Previous analysis of the genome sequence of the archaeon Halobacterium sp. strain NRC-1 has indicated the presence of a sequence homologous to sequences known to encode the glutamate-cysteine ligase GshA. We report here the identification of the gshA gene in the extremely halophilic archaeon Haloferax volcanii and show that H. volcanii gshA directs in vivo the synthesis and accumulation of γGC. We also show that the H. volcanii gene when expressed in an Escherichia coli strain lacking functional GshA is able to restore synthesis of glutathione.Many organisms contain millimolar concentrations of low-molecular-weight thiol compounds that participate in a number of important biological functions involving thiol-disulfide exchanges (7). In particular, they serve to maintain an intracellular reducing environment, to provide reducing power for key reductive enzymes, to combat the effects of oxidative and disulfide stress, and to detoxify xenobiotic compounds (7). Glutathione (GSH), a cysteine-containing tripeptide, l-γ-glutamyl-l-cysteinylglycine, is the best-characterized low-molecular-weight thiol (7, 19, 21). GSH is made in a highly conserved two-step ATP-dependent process by two unrelated peptide bond-forming enzymes (3, 21). The γ-carboxyl group of l-glutamate and the amino group of l-cysteine are ligated by the enzyme glutamylcysteine (GC) ligase EC 6.3.2.2 (GshA, encoded by gshA), which is then condensed with glycine in a reaction catalyzed by GSH synthetase (GshB, encoded by gshB) to form GSH (10, 38). GSH is found primarily in gram-negative bacteria and eukaryotes and only rarely in gram-positive bacteria (26). Fahey and coworkers showed that GSH is absent from the high-GC gram-positive actinomycetes which produce, as the major low-molecular-weight thiol, mycothiol, 1-d-myo-inosityl-2-(N-acetyl-l-cysteinyl)-amido-2-deoxy-α-d-glucopyranoside (13, 26-28, 35). GSH is also absent in Archaea. In Pyrococcus furiosus, coenzyme A SH (CoASH) is the main thiol (11), whereas in Halobacterium salinarum, γGC is the predominant thiol and the organism possesses bis-γGC reductase activity (30, 36). Similarly, Leuconostoc kimchi and Leuconostoc mesenteroides, gram-positive lactic acid bacterial species, were recently found to contain γGC rather than GSH (15). To date, these are the sole procaryotic species reported to naturally produce γGC but not GSH (6, 30). In this report, we describe the identification of the gshA gene in the extremely halophilic archaeon Haloferax volcanii. Copley and Dhillon (6) previously identified, using bioinformatic tools, an open reading frame (ORF) (gene VNG1397C) in Halobacterium sp. strain NRC-1 with limited sequence relatedness to known GshA proteins (6). However, no genetic or biochemical evidence was presented to substantiate their conclusion. Here, we show that Haloferax volcanii strain DS2 (1, 25) contains an ORF that directs in vivo the synthesis and accumulation of γGC. We also show that the H. volcanii ORF, when expressed in Escherichia coli lacking functional GshA, is able to restore synthesis of GSH.     

Analysis of high throughput protein expression in Escherichia coli

The ability to efficiently produce hundreds of proteins in parallel is the most basic requirement of many aspects of proteomics. Overcoming the technical and financial barriers associated with high throughput protein production is essential for the development of an experimental platform to query and browse the protein content of a cell (e.g. protein and antibody arrays). Proteins are inherently different one from another in their physicochemical properties; therefore, no single protocol can be expected to successfully express most of the proteins. Instead of optimizing a protocol to express a specific protein, we used sequence analysis tools to estimate the probability of a specific protein to be expressed successfully using a given protocol, thereby avoiding a priori proteins with a low success probability. A set of 547 proteins, to be used for antibody production and selection, was expressed in Escherichia coli using a high throughput protein production pipeline. Protein properties derived from sequence alone were correlated to successful expression, and general guidelines are given to increase the efficiency of similar pipelines. A second set of 68 proteins was expressed to investigate the link between successful protein expression and inclusion body formation. More proteins were expressed in inclusion bodies; however, the formation of inclusion bodies was not a requirement for successful expression.     

Quantifying the Labeling and the Levels of Plant Cell Wall Precursors Using Ion Chromatography Tandem Mass Spectrometry

The biosynthesis of cell wall polymers involves enormous fluxes through central metabolism that are not fully delineated and whose regulation is poorly understood. We have established and validated a liquid chromatography tandem mass spectrometry method using multiple reaction monitoring mode to separate and quantify the levels of plant cell wall precursors. Target analytes were identified by their parent/daughter ions and retention times. The method allows the quantification of precursors at low picomole quantities with linear responses up to the nanomole quantity range. When applying the technique to Arabidopsis (Arabidopsis thaliana) T87 cell cultures, 16 hexose-phosphates (hexose-Ps) and nucleotide-sugars (NDP-sugars) involved in cell wall biosynthesis were separately quantified. Using hexose-P and NDP-sugar standards, we have shown that hot water extraction allows good recovery of the target metabolites (over 86%). This method is applicable to quantifying the levels of hexose-Ps and NDP-sugars in different plant tissues, such as Arabidopsis T87 cells in culture and fenugreek (Trigonella foenum-graecum) endosperm tissue, showing higher levels of galacto-mannan precursors in fenugreek endosperm. In Arabidopsis cells incubated with [U-¹³C_Fru]sucrose, the method was used to track the labeling pattern in cell wall precursors. As the fragmentation of hexose-Ps and NDP-sugars results in high yields of [PO₃]⁻/or [H₂PO₄]⁻ ions, mass isotopomers can be quantified directly from the intensity of selected tandem mass spectrometry transitions. The ability to directly measure ¹³C labeling in cell wall precursors makes possible metabolic flux analysis of cell wall biosynthesis based on dynamic labeling experiments.Plant cell walls are the most abundant renewable resources (Pauly and Keegstra, 2008a). Much of the current biotechnological research on plant cell wall synthesis involves manipulating these biosynthetic processes to obtain higher concentrations of starches or oil, which show much promise in biofuel production, or to alter cell wall composition for easier breakdown. A detailed knowledge of these processes is essential to understanding and utilizing plant cell wall materials as well as for progress in understanding plant growth and structural development (Pauly and Keegstra, 2008b). However, research into cell wall biosynthesis has been hindered by our limited understanding of the metabolic processes that produce cell walls and particularly their regulation. Progress in this area is limited by the difficulty of differentiating among the compounds involved and of analyzing the fluxes through the biochemical network of wall biosynthesis. Many of the metabolic steps involve isomeric sugars, including hexose-Ps and nucleotide-sugars (NDP-sugars) that serve as direct precursors to plant cell wall biosynthesis. Separate quantification of these sugars has been difficult to achieve.Much of the current research on identifying and differentiating among different metabolic pathways involves the use of chromatography and mass spectrometry (MS; Wolfender et al., 2009). It has been found that liquid chromatography (LC), when linked to a triple quadrupole mass spectrometer (tandem MS [MS/MS]), can be a powerful tool to detect and specifically quantify several classes of metabolic compounds (Allwood and Goodacre, 2010). After initial compound separation by LC, analytes are directed to a triple quadrupole mass spectrometer (MS/MS), where the initial two quadrupoles separate the compounds for detection in the third quadrupole, first by selection of particular mass-to-charge (m/z) ratios of the ionized parent compounds in the first quadrupole, then by fragmentation of the compounds in the second quadrupole (Arrivault et al., 2009). This coupling method of LC-MS/MS to identify compounds has been used with several metabolites involved in plant primary metabolism recently (Cruz et al., 2008; Arrivault et al., 2009).Several studies have reported the separation of phosphorylated metabolites using LC-MS/MS. We are particularly interested in analyzing such compounds because plant cell wall biosynthesis involves a range of phosphorylated precursors, mainly NDP-sugars and hexose-Ps (Feingold and Barber, 1990; Fry 2000, 2004; Seifert, 2004; Somerville et al., 2004; Sharples and Fry, 2007). For example, Huck et al. (2003) and Luo et al. (2007) were able to separate, respectively, six and 28 intracellular metabolites involved in glycolysis, the oxidative pentose-P pathway, and the tricarboxylic acid cycle, some of which are phosphorylated. Bajad et al. (2006) were able to separate a large number of water-soluble cellular metabolites by hydrophilic interaction chromatography, but this method does not appear to separate isomers. Anion exchange chromatography was shown to be effective at separating phosphorylated intermediates involved in glycolysis (van Dam et al., 2002) and in the Calvin cycle (Cruz et al., 2008; Arrivault et al., 2009), especially when coupled with the high specificity and sensitivity of triple quadrupole MS. Moreover, anion-exchange chromatography coupled with MS/MS can be used to determine the mass isotopomer distribution of labeled compounds and Kiefer et al. (2007) were able to quantify isotope abundances in six phosphorylated metabolites in Escherichia coli.However, some of the methods that achieved good separation of four NDP-sugars did not allow quantification by MS/MS because of the eluents used (Räbinä et al., 2001), and none of the methods using coupled LC-MS/MS developed to date separates all or nearly all of the hexose-Ps and NDP-sugars known to be involved in plant cell wall biosynthesis (Turnock and Ferguson, 2007). This presents a special challenge given the fact that many of these sugar compounds are diastereoisomers and ionize similarly in traditional LC-MS/MS methods. Current methods of separating hexose-Ps and NDP-sugars also involve multiple steps of chromatographic and enzymatic separation. In a notable recent study, Sharples and Fry (2007) separated many of the compounds involved in plant cell wall biosynthesis, including hexose-Ps and NDP-sugars, and used radioactive [U-¹⁴C]Fru and [1-³H]Gal as substrates to determine their relative contributions to different cell wall components. The method used in that study involved high-voltage paper electrophoresis separation followed by mild acid hydrolysis and/or phosphatase digestion of different fractions to release neutral hexoses that were then separated by a second paper chromatography procedure. At the cost of considerable effort, this approach allowed eight compounds to be separated. However, neither this nor many of the other approaches used to date appear to have yielded absolute metabolite levels or specific activities in labeling. Metabolic flux analysis requires quantifying these compounds, their fractional and preferably also positional labeling, and the ability to analyze many time point samples. These requirements necessitated the development of a method that can be performed in medium to high throughput and achieves compound separation and quantitation, such as LC-MS/MS, and that also yields detailed labeling information.In this study we have developed and validated a robust and sensitive LC-MS/MS method that successfully allows us to separate and quantify the levels and isotopic labeling of plant cell wall precursors. Using plant tissues from fenugreek (Trigonella foenum-graecum) endosperms and Arabidopsis (Arabidopsis thaliana) cell cultures, 12 hexose-Ps and NDP-sugars known to be involved in plant cell wall biosynthesis were separated and quantified. The direct analysis of intracellular cell wall precursors and their isotopic labeling significantly expands the set of tools for assessing the dynamics and regulation of cell wall biosynthesis, including the potential for dynamic metabolic flux analysis.