Workflow for the Quantification of Soluble and Insoluble Carbohydrates in Soybean Seed.

Soybean seed composition has a profound impact on its market value and commercial use as an important commodity. Increases in oil and protein content have been historically pursued by breeders and genetic engineers; consequently, rapid methods for their quantification are well established. The interest in complete carbohydrate profiles in mature seeds, on the other hand, has recently increased due to numerous attempts to redirect carbohydrates into oil and protein or to offer specialty seed with a specific sugar profile to meet animal nutritional requirements. In this work, a sequential protocol for quantifying reserve and structural carbohydrates in soybean seed was developed and validated. Through this procedure, the concentrations of soluble sugars, sugar alcohols, starch, hemicellulose, and crystalline cellulose can be determined in successive steps from the same starting material using colorimetric assays, LC-MS/MS, and GC-MS. The entire workflow was evaluated using internal standards to estimate the recovery efficiency. Finally, it was successfully applied to eight soybean genotypes harvested from two locations, and the resulting correlations of carbohydrate and oil or protein are presented. This methodology has the potential not only to guide soybean cultivar optimization processes but also to be expanded to other crops with only slight modifications.

The gluconate shunt is an alternative route for directing glucose into the pentose phosphate pathway in fission yeast.

Glycolysis and the pentose phosphate pathway both play a central role in the degradation of glucose in all domains of life. Another metabolic route that can facilitate glucose breakdown is the gluconate shunt. In this shunt glucose dehydrogenase and gluconate kinase catalyze the two-step conversion of glucose into the pentose phosphate pathway intermediate 6-phosphogluconate. Despite the presence of these enzymes in many organisms, their only established role is in the production of 6-phosphogluconate for the Entner-Doudoroff pathway. In this report we performed metabolic profiling on a strain of Schizosaccharomyces pombe lacking the zinc-responsive transcriptional repressor Loz1 with the goal of identifying metabolic pathways that were altered by cellular zinc status. This profiling revealed that loz1Δ cells accumulate higher levels of gluconate. We show that the altered gluconate levels in loz1Δ cells result from increased expression of gcd1 By analyzing the activity of recombinant Gcd1 in vitro and by measuring gluconate levels in strains lacking enzymes of the gluconate shunt we demonstrate that Gcd1 encodes a novel NADP+-dependent glucose dehydrogenase that acts in a pathway with the Idn1 gluconate kinase. We also find that cells lacking gcd1 and zwf1, which encode the first enzyme in the pentose phosphate pathway, have a more severe growth phenotype than cells lacking zwf1 We propose that in S. pombe Gcd1 and Idn1 act together to shunt glucose into the pentose phosphate pathway, creating an alternative route for directing glucose into the pentose phosphate pathway that bypasses hexokinase and the rate-limiting enzyme glucose-6-phosphate dehydrogenase.

A liquid chromatography tandem mass spectroscopy approach for quantification of protein methylation stoichiometry.

Post-translational modifications are biologically important and wide-spread modulators of protein function. Although methods for detecting the presence of specific modifications are becoming established, approaches for quantifying their mol modification/mol protein stoichiometry are less well developed. Here we introduce a ratiometric, label-free, targeted liquid chromatography tandem mass spectroscopy-based method for estimating Lys and Arg methylation stoichiometry on post-translationally modified proteins. Methylated Lys and Arg were detected with limits of quantification at low fmol and with linearity extending from 20 to 5000 fmol. This level of sensitivity allowed estimation of methylation stoichiometry from microgram quantities of various proteins, including those derived from either recombinant or tissue sources. The method also disaggregated total methylation stoichiometry into its elementary mono-, di-, and tri-methylated residue components. In addition to being compatible with kinetic experiments of protein methylation, the approach will be especially useful for characterizing methylation states of proteins isolated from cells and tissues.

Identification of multiple lipid genes with modifications in expression and sequence associated with the evolution of hydroxy fatty acid accumulation in Physaria fendleri.

Two Brassicaceae species, Physaria fendleri and Camelina sativa, are genetically very closely related to each other and to Arabidopsis thaliana. Physaria fendleri seeds contain over 50% hydroxy fatty acids (HFAs), while Camelina sativa and Arabidopsis do not accumulate HFAs. To better understand how plants evolved new biochemical pathways with the capacity to accumulate high levels of unusual fatty acids, transcript expression and protein sequences of developing seeds of Physaria fendleri, wild-type Camelina sativa, and Camelina sativa expressing a castor bean (Ricinus communis) hydroxylase were analyzed. A number of potential evolutionary adaptations within lipid metabolism that probably enhance HFA production and accumulation in Physaria fendleri, and, in their absence, limit accumulation in transgenic tissues were revealed. These adaptations occurred in at least 20 genes within several lipid pathways from the onset of fatty acid synthesis and its regulation to the assembly of triacylglycerols. Lipid genes of Physaria fendleri appear to have co-evolved through modulation of transcriptional abundances and alterations within protein sequences. Only a handful of genes showed evidence for sequence adaptation through gene duplication. Collectively, these evolutionary changes probably occurred to minimize deleterious effects of high HFA amounts and/or to enhance accumulation for physiological advantage. These results shed light on the evolution of pathways for novel fatty acid production in seeds, help explain some of the current limitations to accumulation of HFAs in transgenic plants, and may provide improved strategies for future engineering of their production.

Dynamic metabolic flux analysis of plant cell wall synthesis.

The regulation of plant cell wall synthesis pathways remains poorly understood. This has become a bottleneck in designing bioenergy crops. The goal of this study was to analyze the regulation of plant cell wall precursor metabolism using metabolic flux analysis based on dynamic labeling experiments. Arabidopsis T87 cells were cultured heterotrophically with (13)C labeled sucrose. The time course of ¹³C labeling patterns in cell wall precursors and related sugar phosphates was monitored using liquid chromatography tandem mass spectrometry until steady state labeling was reached. A kinetic model based on mass action reaction mechanisms was developed to simulate the carbon flow in the cell wall synthesis network. The kinetic parameters of the model were determined by fitting the model to the labeling time course data, cell wall composition, and synthesis rates. A metabolic control analysis was performed to predict metabolic regulations that may improve plant biomass composition for biofuel production. Our results describe the routes and rates of carbon flow from sucrose to cell wall precursors. We found that sucrose invertase is responsible for the entry of sucrose into metabolism and UDP-glucose-4-epimerase plays a dominant role in UDP-Gal synthesis in heterotrophic Aradidopsis cells under aerobic conditions. We also predicted reactions that exert strong regulatory influence over carbon flow to cell wall synthesis and its composition.

Deep EST profiling of developing fenugreek endosperm to investigate galactomannan biosynthesis and its regulation.

Galactomannans are hemicellulosic polysaccharides composed of a (1 â†’ 4)-linked ß-D-mannan backbone substituted with single-unit (1 â†’ 6)-α-linked D-galactosyl residues. Developing fenugreek (Trigonella foenum-graecum) seeds are known to accumulate large quantities of galactomannans in the endosperm, and were thus used here as a model system to better understand galactomannan biosynthesis and its regulation. We first verified the specific deposition of galactomannans in developing endosperms and determined that active accumulation occurred from 25 to 38 days post anthesis (DPA) under our growth conditions. We then examined the expression levels during seed development of ManS and GMGT, two genes encoding backbone and side chain synthetic enzymes. Based on transcript accumulation dynamics for ManS and GMGT, cDNA libraries were constructed using RNA isolated from endosperms at four ages corresponding to before, at the beginning of, and during active galactomannan deposition. DNA from these libraries was sequenced using the 454 sequencing technology to yield a total of 1.5 million expressed sequence tags (ESTs). Through analysis of the EST profiling data, we identified genes known to be involved in galactomannan biosynthesis, as well as new genes that may be involved in this process, and proposed a model for the flow of carbon from sucrose to galactomannans. Measurement of in vitro ManS and GMGT activities and analysis of sugar phosphate and nucleotide sugar levels in the endosperms of developing fenugreek seeds provided data consistent with this model. In vitro enzymatic assays also revealed that the ManS enzyme from fenugreek endosperm preferentially used GDP-mannose as the substrate for the backbone synthesis.

Central metabolic fluxes in the endosperm of developing maize seeds and their implications for metabolic engineering.

¹4C labeling experiments performed with kernel cultures showed that developing maize endosperm is more efficient than other non-photosynthetic tissues such as sunflower and maize embryos at converting maternally supplied substrates into biomass. To characterize the metabolic fluxes in endosperm, maize kernels were labeled to isotopic steady state using ¹³C-labeled glucose. The resultant labeling in free metabolites and biomass was analyzed by NMR and GC-MS. After taking into account the labeling of substrates supplied by the metabolically active cob, the fluxes through central metabolism were quantified by computer-aided modeling. The flux map indicates that 51-69% of the ATP produced is used for biomass synthesis and up to 47% is expended in substrate cycling. These findings point to potential engineering targets for improving yield and increasing oil contents by, respectively, reducing substrate cycling and increasing the commitment of plastidic carbon into fatty acid synthesis at the level of pyruvate kinase.

Synergy between (13)C-metabolic flux analysis and flux balance analysis for understanding metabolic adaptation to anaerobiosis in E. coli.

Genome-based Flux Balance Analysis (FBA) and steady-state isotopic-labeling-based Metabolic Flux Analysis (MFA) are complimentary approaches to predicting and measuring the operation and regulation of metabolic networks. Here, genome-derived models of Escherichia coli (E. coli) metabolism were used for FBA and ¹³C-MFA analyses of aerobic and anaerobic growths of wild-type E. coli (K-12 MG1655) cells. Validated MFA flux maps reveal that the fraction of maintenance ATP consumption in total ATP production is about 14% higher under anaerobic (51.1%) than aerobic conditions (37.2%). FBA revealed that an increased ATP utilization is consumed by ATP synthase to secrete protons from fermentation. The TCA cycle is shown to be incomplete in aerobically growing cells and submaximal growth is due to limited oxidative phosphorylation. An FBA was successful in predicting product secretion rates in aerobic culture if both glucose and oxygen uptake measurement were constrained, but the most-frequently predicted values of internal fluxes yielded from sampling the feasible space differ substantially from MFA-derived fluxes.

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-(13)C(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 [PO(3)](-)/or [H(2)PO(4)](-) ions, mass isotopomers can be quantified directly from the intensity of selected tandem mass spectrometry transitions. The ability to directly measure (13)C labeling in cell wall precursors makes possible metabolic flux analysis of cell wall biosynthesis based on dynamic labeling experiments.

Liquid Chromatography Tandem Mass Spectrometry Quantification of 13C-Labeling in Sugars.

Subcellular compartmentation has been challenging in plant 13C-metabolic flux analysis. Indeed, plant cells are highly compartmented: they contain vacuoles and plastids in addition to the regular organelles found in other eukaryotes. The distinction of reactions between compartments is possible when metabolites are synthesized in a particular compartment or by a unique pathway. Sucrose is an example of such a metabolite: it is specifically produced in the cytosol from glucose 6-phosphate (G6P) and fructose 6-phosphate (F6P). Therefore, determining the 13C-labeling in the fructosyl and glucosyl moieties of sucrose directly informs about the labeling of cytosolic F6P and G6P, respectively. To date, the most commonly used method to monitor sucrose labeling is by nuclear magnetic resonance, which requires substantial amounts of biological sample. This study describes a new methodology that accurately measures the labeling in free sugars using liquid chromatography tandem mass spectrometry (LC-MS/MS). For this purpose, maize embryos were pulsed with [U-13C]-fructose, intracellular sugars were extracted, and their time-course labeling was analyzed by LC-MS/MS. Additionally, extracts were enzymatically treated with hexokinase to remove the soluble hexoses, and then invertase to cleave sucrose into fructose and glucose. Finally, the labeling in the glucosyl and fructosyl moieties of sucrose was determined by LC-MS/MS.

Metabolomics as an Emerging Tool for the Study of Plant-Pathogen Interactions.

Plants defend themselves from most microbial attacks via mechanisms including cell wall fortification, production of antimicrobial compounds, and generation of reactive oxygen species. Successful pathogens overcome these host defenses, as well as obtain nutrients from the host. Perturbations of plant metabolism play a central role in determining the outcome of attempted infections. Metabolomic analyses, for example between healthy, newly infected and diseased or resistant plants, have the potential to reveal perturbations to signaling or output pathways with key roles in determining the outcome of a plant-microbe interaction. However, application of this -omic and its tools in plant pathology studies is lagging relative to genomic and transcriptomic methods. Thus, it is imperative to bring the power of metabolomics to bear on the study of plant resistance/susceptibility. This review discusses metabolomics studies that link changes in primary or specialized metabolism to the defense responses of plants against bacterial, fungal, nematode, and viral pathogens. Also examined are cases where metabolomics unveils virulence mechanisms used by pathogens. Finally, how integrating metabolomics with other -omics can advance plant pathology research is discussed.

Differential Metabolic Responses Caused by the Most Important Insect Pest of Coffee Worldwide, the Coffee Berry Borer (Hypothenemus hampei).

The world's coffee supply is threatened by the coffee berry borer, Hypothenemus hampei, the most destructive pest affecting coffee production and quality. This study hypothesized that coffee berry borer infestation induces distinct metabolic responses in the green coffee seeds of Coffea arabica and Coffea canephora (robusta). A targeted metabolomics approach was conducted using liquid chromatography tandem mass spectrometry to quantify intracellular metabolites in infested and uninfested arabica and robusta green seeds. In parallel, the seed biomass content and composition were assessed for the same conditions. Coffee berry borer attack induced increases in the levels of chlorogenic acids in arabica seeds, whereas organic acids and sugar alcohols were more abundant in infested robusta seeds. Most importantly, a set of compounds was identified as biomarkers differentiating the metabolic response of these taxa to the coffee berry borer.

Quantification of Tau Protein Lysine Methylation in Aging and Alzheimer's Disease.

Tau is a microtubule-associated protein that normally interacts in monomeric form with the neuronal cytoskeleton. In Alzheimer's disease, however, it aggregates to form the structural component of neurofibrillary lesions. The transformation is controlled in part by age- and disease-associated post-translational modifications. Recently we reported that tau isolated from cognitively normal human brain was methylated on lysine residues, and that high-stoichiometry methylation depressed tau aggregation propensity in vitro. However, whether methylation stoichiometry reached levels needed to influence aggregation propensity in human brain was unknown. Here we address this problem using liquid chromatography-tandem mass spectrometry approaches and human-derived tau samples. Results revealed that lysine methylation was present in soluble tau isolated from cognitively normal elderly cases at multiple sites that only partially overlapped with the distributions reported for cognitively normal middle aged and AD cohorts, and that the quality of methylation shifted from predominantly dimethyl-lysine to monomethyl-lysine with aging and disease. However, bulk mol methylation/mol tau stoichiometries never exceeded 1 mol methyl group/mol tau protein. We conclude that lysine methylation is a physiological post-translational modification of tau protein that changes qualitatively with aging and disease, and that pharmacological elevation of tau methylation may provide a means for protecting against pathological tau aggregation.

High-throughput quantification of the levels and labeling abundance of free amino acids by liquid chromatography tandem mass spectrometry.

Accurate assessment of mass isotopomer distributions (MIDs) of intracellular metabolites, such as free amino acids (AAs), is crucial for quantifying in vivo fluxes. To date, the majority of studies that measured AA MIDs have relied on the analysis of proteinogenic rather than free AAs by: i) GC-MS, which involved cumbersome process of derivatization, or ii) NMR, which requires large quantities of biological sample. In this work, the development and validation of a high-throughput LC-MS/MS method allowing the quantification of the levels and labeling of free AAs is described. Sensitivity in the order of the femtomol was achieved using multiple reaction monitoring mode (MRM). The MIDs of all free AAs were assessed without the need of derivatization, and were validated (except for Trp) on a mixture of unlabeled AA standards. Finally, this method was applied to the determination of the 13C-labeling abundance in free AAs extracted from maize embryos cultured with 13C-glutamine or 13C-glucose. Although Cys was below the limit of detection in these biological samples, the MIDs of a total of 18 free AAs were successfully determined. Due to the increased application of tandem mass spectrometry for 13C-Metabolic Flux Analysis, this novel method will enable the assessment of more complete and accurate labeling information of intracellular AAs, and therefore a better definition of the fluxes.

Identification of biochemical features of defective Coffea arabica L. beans.

Coffee organoleptic properties are based in part on the quality and chemical composition of coffee beans. The presence of defective beans during processing and roasting contribute to off flavors and reduce overall cup quality. A multipronged approach was undertaken to identify specific biochemical markers for defective beans. To this end, beans were split into defective and non-defective fractions and biochemically profiled in both green and roasted states. A set of 17 compounds in green beans, including organic acids, amino acids and reducing sugars; and 35 compounds in roasted beans, dominated by volatile compounds, organic acids, sugars and sugar alcohols, were sufficient to separate the defective and non-defective fractions. Unsorted coffee was examined for the presence of the biochemical markers to test their utility in detecting defective beans. Although the green coffee marker compounds were found in all fractions, three of the roasted coffee marker compounds (1-methylpyrrole, 5-methyl- 2-furfurylfuran, and 2-methylfuran) were uniquely present in defective fractions.

Carbon conversion efficiency and central metabolic fluxes in developing sunflower (Helianthus annuus L.) embryos.

The efficiency with which developing sunflower embryos convert substrates into seed storage reserves was determined by labeling embryos with [U-(14)C6]glucose or [U-(14)C5]glutamine and measuring their conversion to CO2, oil, protein and other biomass compounds. The average carbon conversion efficiency was 50%, which contrasts with a value of over 80% previously observed in Brassica napus embryos (Goffman et al., 2005), in which light and the RuBisCO bypass pathway allow more efficient conversion of hexose to oil. Labeling levels after incubating sunflower embryos with [U-(14)C4]malate indicated that some carbon from malate enters the plastidic compartment and contributes to oil synthesis. To test this and to map the underlying pattern of metabolic fluxes, separate experiments were carried out in which embryos were labeled to isotopic steady state using [1-(13)C1]glucose, [2-(13)C1]glucose, or [U-(13)C5]glutamine. The resultant labeling in sugars, starch, fatty acids and amino acids was analyzed by NMR and GC-MS. The fluxes through intermediary metabolism were then quantified by computer-aided modeling. The resulting flux map accounted well for the labeling data, was in good agreement with the observed carbon efficiency, and was further validated by testing for agreement with gas exchange measurements. The map shows that the influx of malate into oil is low and that flux through futile cycles (wasting ATP) is low, which contrasts with the high rates previously determined for growing root tips and heterotrophic cell cultures.

A gene from the cellulose synthase-like C family encodes a beta-1,4 glucan synthase.

Despite the central role of xyloglucan (XyG) in plant cell wall structure and function, important details of its biosynthesis are not understood. To identify the gene(s) responsible for synthesizing the beta-1,4 glucan backbone of XyG, we exploited a property of nasturtium (Tropaeolum majus) seed development. During the last stages of nasturtium seed maturation, a large amount of XyG is deposited as a reserve polysaccharide. A cDNA library was produced from mRNA isolated during the deposition of XyG, and partial sequences of 10,000 cDNA clones were determined. A single member of the C subfamily from the large family of cellulose synthase-like (CSL) genes was found to be overrepresented in the cDNA library. Heterologous expression of this gene in the yeast Pichia pastoris resulted in the production of a beta-1,4 glucan, confirming that the CSLC protein has glucan synthase activity. The Arabidopsis CSLC4 gene, which is the gene with the highest sequence similarity to the nasturtium CSL gene, is coordinately expressed with other genes involved in XyG biosynthesis. These and other observations provide a compelling case that the CSLC gene family encode proteins that synthesize the XyG backbone.

Light enables a very high efficiency of carbon storage in developing embryos of rapeseed.

The conversion of photosynthate to seed storage reserves is crucial to plant fitness and agricultural production, yet quantitative information about the efficiency of this process is lacking. To measure metabolic efficiency in developing seeds, rapeseed (Brassica napus) embryos were cultured in media in which all carbon sources were [U-14C]-labeled and their conversion into CO2, oil, protein, and other biomass was determined. The conversion efficiency of the supplied carbon into seed storage reserves was very high. When provided with 0, 50, or 150 micromol m(-2) s(-1) light, the proportion of carbon taken up by embryos that was recovered in biomass was 60% to 64%, 77% to 86%, and 85% to 95%, respectively. Light not only improved the efficiency of carbon storage, but also increased the growth rate, the proportion of 14C recovered in oil relative to protein, and the fixation of external 14CO2 into biomass. Embryos grown at 50 micromol m(-2) s(-1) in the presence of 5 microM 1,1-dimethyl-3-(3,4-dichlorophenyl) urea (an inhibitor of photosystem II) were reduced in total biomass and oil synthesis by 3.2-fold and 2.8-fold, respectively, to the levels observed in the dark. To explore if the reduced growth and carbon conversion efficiency in dark were related to oxygen supplied by photosystem II, embryos and siliques were cultured with increased oxygen. The carbon conversion efficiency of embryos remained unchanged when oxygen levels were increased 3-fold. Increasing the O2 levels surrounding siliques from 21% to 60% did not increase oil synthesis rates either at 1,000 micromol m(-2) s(-1) or in the dark. We conclude that light increases the growth, efficiency of carbon storage, and oil synthesis in developing rapeseed embryos primarily by providing reductant and/or ATP.