Correction to: Identification of an intracellular metabolic signature impairing beta cell function in the rat beta cell line INS-1E and human islets.

As part of an institutional investigation by University of Bremen, the work carried out by Kathrin Maedler's laboratory has been reviewed.

Milk metabolites and their genetic variability.

The composition of milk is crucial to evaluate milk performance and quality measures. Milk components partly contribute to breeding scores, and they can be assessed to judge metabolic and energy status of the cow as well as to serve as predictive markers for diseases. In addition to the milk composition measures (e.g., fat, protein, lactose) traditionally recorded during milk performance test via infrared spectroscopy, novel techniques, such as gas chromatography-mass spectrometry, allow for a further analysis of milk into its metabolic components. Gas chromatography-mass spectrometry is suitable for measuring several hundred metabolites with high throughput, and thus it is applicable to study sources of genetic and nongenetic variation of milk metabolites in dairy cows. Heritability and mode of inheritance of metabolite measurements were studied in a linear mixed model approach including expected (pedigree) and realized (genomic) relationship between animals. The genetic variability of 190 milk metabolite intensities was analyzed from 1,295 cows held on 18 farms in Mecklenburg-Western Pomerania, Germany. Besides extensive pedigree information, genotypic data comprising 37,180 single nucleotide polymorphism markers were available. Goodness of fit and significance of genetic variance components based on likelihood ratio tests were investigated with a full model, including marker- and pedigree-based genetic effects. Broad-sense heritability varied from zero to 0.699, with a median of 0.125. Significant additive genetic variance was observed for highly heritable metabolites, but dominance variance was not significantly present. As some metabolites are particularly favorable for human nutrition, for instance, future research should address the identification of locus-specific genetic effects and investigate metabolites as the molecular basis of traditional milk performance test traits.

Investigating associations between milk metabolite profiles and milk traits of Holstein cows.

In the field of dairy cattle research, it is of great interest to improve the detection and prevention of diseases (e.g., mastitis and ketosis) and monitor specific traits related to the state of health and management. During the standard milk performance test, traditional milk traits are monitored, and quality and quantity are screened. In addition to the standard test, it is also now possible to analyze milk metabolites in a high-throughput manner and to consider them in connection with milk traits to identify functionally important metabolites that can also serve as biomarker candidates. We present a study in which 190 milk metabolites and 14 milk traits of 1,305 Holstein cows on 18 commercial farms were investigated to characterize interrelations of milk metabolites between each other, to milk traits from the milk standard performance test, and to influencing factors such as farm and sire effect (half-sib structure). The effect of influencing factors (e.g., farm) varied among metabolites and traditional milk traits. The investigations of associations between metabolites and milk traits revealed groups of metabolites that show, for example, positive correlations to protein and casein, and negative correlations to lactose and pH. On the other hand, groups of metabolites jointly associated with the investigated milk traits can be identified and functionally discussed. To enable a multivariate investigation, 2 machine learning methods were applied to detect important metabolites that are highly correlated with the investigated traditional milk traits. For somatic cell score, uracil, lactic acid, and 9 other important metabolites were detected. Lactic acid has already been proposed as a biomarker candidate for mastitis in the recent literature. In conclusion, we found sets of metabolites eligible to predict milk traits, enabling the analysis of milk traits from a metabolic perspective and discussion of the possible functional background for some of the detected associations.

Identification of an intracellular metabolic signature impairing beta cell function in the rat beta cell line INS-1E and human islets.

AIMS/HYPOTHESIS: Chronic hyperglycaemia promotes the progressive failure of pancreatic beta cells in patients with type 2 diabetes mellitus, a clinically highly relevant phenomenon known as glucotoxicity. The intracellular metabolic consequences of a chronically high availability of glucose in beta cells are, as yet, poorly understood in its full complexity. METHODS: An unbiased metabolite profiling analysis (GC-time-of-flight-MS) was used to identify the time course of core metabolite patterns in rat beta cell line INS-1E during exposure to high glucose concentrations and its relation to insulin expression. RESULTS: We report here that pentose phosphate pathway (PPP) metabolites accumulate remarkably during chronic but not acute glucose treatment, indicating altered processing of glucose through the pentose phosphate pathway. Subsequent functional studies in INS-1E cells and human islets revealed that a disturbance in this pathway contributes to decreases in insulin gene expression and a lack of glucose-stimulated insulin secretion. These effects were found to depend on the activation of extracellular-regulated-kinase (ERK1/2). Long-term inhibition of 6-phosphogluconic acid dehydrogenase resulted in accumulation of PPP metabolites, induced ERK1/2 activation independently of high glucose and impaired beta cell function. In turn, inhibition of ERK1/2 overstimulation during chronic glucose exposure partly inhibited metabolite accumulation and restored beta cell function. CONCLUSIONS/INTERPRETATION: Based on unbiased metabolite analyses, the data presented here provide novel targets, namely the inhibition of PPP metabolite accumulation towards the therapeutic goal to preserve and potentially improve beta cell function in diabetes.

Inhibition of a ubiquitously expressed pectin methyl esterase in Solanum tuberosum L. affects plant growth, leaf growth polarity, and ion partitioning.

Two pectin methyl esterases (PMEs; EC 3.1.1.11) from Solanum tuberosum were isolated and their expression characterised. One partial clone ( pest1) was expressed in leaves and fruit tissue, while pest2 was a functional full-length clone and was expressed ubiquitously, with a preference for aerial organs. Potato plants were transformed with a chimeric antisense construct that was designed to simultaneously inhibit pest1 and pest2 transcript accumulation; however, reduction of mRNA levels was confined to pest2. The decrease in pest2 transcript was accompanied by up to 50% inhibition of total PME activity, which was probably due to the reduction of only one PME isoform. PME inhibition affected plant development as reflected by smaller stem elongation rates of selected transformants when compared with control plants, leading to a reduction in height throughout the entire course of development. Expansion rates of young developing leaves were measured simultaneously by two displacement transducers in the direction of the leaf tip (proximal-distal axis) and in the perpendicular direction (medial-lateral axis). Significant differences in leaf growth patterns were detected between wild-type and transgenic plants. We suggest that these visual phenotypes could be correlated with modifications of ion accumulation and partitioning within the transgenic plants. The ion-binding capacities of cell walls from PME-inhibited plants were specifically modified as they preferentially bound more sodium, but less potassium and calcium. X-ray microanalysis also indicated an increase in the concentration of several ions within the leaf apoplast of transgenic plants. Moreover, quantification of the total content of major cations revealed differences specific for a given element between the leaves of PME-inhibited and wild-type plants. Reduced growth rates might also be due to effects of PME inhibition on pectin metabolism, predominantly illustrated by an accumulation of galacturonic acid over other cell wall components.

High-resolution metabolic phenotyping of genetically and environmentally diverse potato tuber systems. Identification of phenocopies.

We conducted a comprehensive metabolic phenotyping of potato (Solanum tuberosum L. cv Desiree) tuber tissue that had been modified either by transgenesis or exposure to different environmental conditions using a recently developed gas chromatography-mass spectrometry profiling protocol. Applying this technique, we were able to identify and quantify the major constituent metabolites of the potato tuber within a single chromatographic run. The plant systems that we selected to profile were tuber discs incubated in varying concentrations of fructose, sucrose, and mannitol and transgenic plants impaired in their starch biosynthesis. The resultant profiles were then compared, first at the level of individual metabolites and then using the statistical tools hierarchical cluster analysis and principal component analysis. These tools allowed us to assign clusters to the individual plant systems and to determine relative distances between these clusters; furthermore, analyzing the loadings of these analyses enabled identification of the most important metabolites in the definition of these clusters. The metabolic profiles of the sugar-fed discs were dramatically different from the wild-type steady-state values. When these profiles were compared with one another and also with those we assessed in previous studies, however, we were able to evaluate potential phenocopies. These comparisons highlight the importance of such an approach in the functional and qualitative assessment of diverse systems to gain insights into important mediators of metabolism.

Antisense inhibition of threonine synthase leads to high methionine content in transgenic potato plants.

Methionine (Met) and threonine (Thr) are members of the aspartate family of amino acids. In plants, their biosynthetic pathways diverge at the level of O-phosphohomo-serine (Ser). The enzymes cystathionine gamma-synthase and Thr synthase (TS) compete for the common substrate O-phosphohomo-Ser with the notable feature that plant TS is activated through S-adenosyl-Met, a metabolite derived from Met. To investigate the regulation of this branch point, we engineered TS antisense potato (Solanum tuberosum cv Désirée) plants using the constitutive cauliflower mosaic virus 35S promoter. In leaf tissues, these transgenics exhibit a reduction of TS activity down to 6% of wild-type levels. Thr levels are reduced to 45% wild-type controls, whereas Met levels increase up to 239-fold depending on the transgenic line and environmental conditions. Increased levels of homo-Ser and homo-cysteine indicate increased carbon allocation into the aspartate pathway. In contrast to findings in Arabidopsis, increased Met content has no detectable effect on mRNA or protein levels or on the enzymatic activity of cystathionine gamma-synthase in potato. Tubers of TS antisense potato plants contain a Met level increased by a factor of 30 and no reduction in Thr. These plants offer a major biotechnological advance toward the development of crop plants with improved nutritional quality.

Analysis of the compartmentation of glycolytic intermediates, nucleotides, sugars, organic acids, amino acids, and sugar alcohols in potato tubers using a nonaqueous fractionation method.

The compartmentation of metabolism in heterotrophic plant tissues is poorly understood due to the lack of data on metabolite distributions and fluxes between subcellular organelles. The main reason for this is the lack of suitable experimental methods with which intracellular metabolism can be measured. Here, we describe a nonaqueous fractionation method that allows the subcellular distributions of metabolites in developing potato (Solanum tuberosum L. cv Desiree) tubers to be calculated. In addition, we have coupled this fractionation method to a recently described gas chromatography-mass spectrometry procedure that allows the measurement of a wide range of small metabolites. To calculate the subcellular metabolite concentrations, we have analyzed organelle volumes in growing potato tubers using electron microscopy. The relative volume distributions in tubers are very similar to the ones for source leaves. More than 60% of most sugars, sugar alcohols, organic acids, and amino acids were found in the vacuole, although the concentrations of these metabolites is often higher in the cytosol. Significant amounts of the substrates for starch biosynthesis, hexose phosphates, and ATP were found in the plastid. However, pyrophosphate was located almost exclusively in the cytosol. Calculation of the mass action ratios of sucrose synthase, UDP-glucose pyrophosphorylase, phosphoglucosisomerase, and phosphoglucomutase indicate that these enzymes are close to equilibrium in developing potato tubers. However, due to the low plastidic pyrophosphate concentration, the reaction catalyzed by ADP-glucose pyrophosphorylase was estimated to be far removed from equilibrium.

Identification and quantification of catecholamines in potato plants (Solanum tuberosum) by GC-MS.

Dopamine, norepinephrine, and normetanephrine were identified by GC-MS in potato (Solanum tuberosum L.) plants, the latter was new for plants. The highest amount of catecholamines was found in leaves. A developmental stage dependent variation in potato leaf catecholamines accumulation was also observed with highest level in third leaves. Catecholamine contents decrease during cold storage of tubers to undetectable levels. Mechanical wounding of leaves led to a small increase in the level of catecholamines investigated.

The contribution of plastidial phosphoglucomutase to the control of starch synthesis within the potato tuber.

The aim of this work was to evaluate the extent to which plastidial phosphoglucomutase (PGM) activity controls starch synthesis within potato (Solanum tuberosum L. cv. Desirée) tubers. The reduction in the activity of plastidial PGM led to both a correlative reduction in starch accumulation and an increased sucrose accumulation. The control coefficient of plastidial PGM on the accumulation of starch was estimated to approximate 0.24. The fluxes of carbohydrate metabolism were measured by investigating the metabolism of [U-14C]glucose in tuber discs from wild-type and transgenic plants. In tuber discs the control coefficient of plastidial PGM over starch synthesis was estimated as 0.36, indicating that this enzyme exerts considerable control over starch synthesis within the potato tuber.

Approaches towards understanding methionine biosynthesis in higher plants.

Plants are able to synthesise all amino acids essential for human and animal nutrition. Because the concentrations of some of these dietary constituents, especially methionine, lysine, and threonine, are often low in edible plant sources, research is being performed to understand the physiological, biochemical, and molecular mechanisms that contribute to their transport, synthesis and accumulation in plants. This knowledge can be used to develop strategies allowing a manipulation of crop plants, eventually improving their nutritional quality. This article is intended to serve two purposes. The first is to provide a brief review on the physiology of methionine synthesis in higher plants. The second is to highlight some recent findings linked to the metabolism of methionine in plants due to its regulatory influence on the aspartate pathway and its implication in plant growth. This information can be used to develop strategies to improve methionine content of plants and to provide crops with a higher nutritional value.

Manipulation of thiol contents in plants.

As sulfur constitutes one of the macronutrients necessary for the plant life cycle, sulfur uptake and assimilation in higher plants is one of the crucial factors determining plant growth and vigour, crop yield and even resistance to pests and stresses. Inorganic sulfate is mostly taken up as sulfate from the soil through the root system or to a lesser extent as volatile sulfur compounds from the air. In a cascade of enzymatic steps inorganic sulfur is converted to the nutritionally important sulfur-containing amino acids cysteine and methionine (Hell, 1997; Hell and Rennenberg, 1998; Saito, 1999). Sulfate uptake and allocation between plant organs or within the cell is mediated by specific transporters localised in plant membranes. Several functionally different sulfate transporters have to be postulated and have been already cloned from a number of plant species (Clarkson et al., 1993; Hawkesford and Smith, 1997; Takahashi et al., 1997; Yamaguchi, 1997). Following import into the plant and transport to the final site of reduction, the plastid, the chemically relatively inert sulfate molecule is activated through binding to ATP forming adenosine-5'-phosphosulfate (APS). This enzymatic step is controlled through the enzyme ATP-sulfurylase (ATP-S). APS can be further phosphorylated to form 3'-phosphoadenosine-5'-phosphosulfate (PAPS) which serves as sulfate donor for the formation of sulfate esters such as the biosynthesis of sulfolipids (Schmidt and Jäger, 1992). However, most of the APS is reduced to sulfide through the enzymes APS-reductase (APR) and sulfite reductase (SIR). The carbon backbone of cysteine is provided through serine, thus directly coupling photosynthetic processes and nitrogen metabolism to sulfur assimilation. L-serine is activated by serine acetyltransferase (SAT) through the transfer to an acetyl-group from acetyl coenzyme A to form O-acetyl-L-serine (OAS) which is then sulhydrylated using sulfide through the enzyme O-acetyl-L-serine thiol lyase (OAS-TL) forming cysteine. Cysteine is the central precursor of all organic molecules containing reduced sulfur ranging from the amino acid methionine to peptides as glutathione or phytochelatines, proteines, vitamines, cofactors as SAM and hormones. Cysteine and derived metabolites display essential roles within plant metabolism such as protein stabilisation through disulfide bridges, stress tolerance to active oxygen species and metals, cofactors for enzymatic reactions as e.g. SAM as major methylgroup donor and plant development and signalling through the volatile hormone ethylene. Cysteine and other metabolites carrying free sulfhydryl groups are commonly termed thioles (confer Fig. 1). The physiological control of the sulfate reduction pathway in higher plants is still not completely understood in all details. The objective of this paper is to summarise the available data on the molecular analysis and control of cysteine biosynthesis in plants, and to discuss potentials for manipulating the pathway using transgenic approaches.

Acceleration of potato tuber sprouting by the expression of a bacterial pyrophosphatase.

Potato is a globally important crop. Unfortunately, potato farming is plagued with problems associated with the sprouting behavior of seed tubers. The data presented here demonstrate that using transgenic technology can influence this behavior. Transgenic tubers cytosolically expressing an inorganic pyrophosphatase gene derived from Escherichia coli under the control of the tuber-specific patatin promoter display significantly accelerated sprouting. The period of presprouting dormancy for transgenic tubers planted immediately after harvest is reduced by six to seven weeks when compared to wild-type tubers. This study demonstrates a method with which to regulate dormancy, an important aspect of potato crop management.

Metabolic profiling allows comprehensive phenotyping of genetically or environmentally modified plant systems.

Metabolic profiling using gas chromatography-mass spectrometry technologies is a technique whose potential in the field of functional genomics is largely untapped. To demonstrate the general usefulness of this technique, we applied to diverse plant genotypes a recently developed profiling protocol that allows detection of a wide range of hydrophilic metabolites within a single chromatographic run. For this purpose, we chose four independent potato genotypes characterized by modifications in sucrose metabolism. Using data-mining tools, including hierarchical cluster analysis and principle component analysis, we were able to assign clusters to the individual plant systems and to determine relative distances between these clusters. Extraction analysis allowed identification of the most important components of these clusters. Furthermore, correlation analysis revealed close linkages between a broad spectrum of metabolites. In a second, complementary approach, we subjected wild-type potato tissue to environmental manipulations. The metabolic profiles from these experiments were compared with the data sets obtained for the transgenic systems, thus illustrating the potential of metabolic profiling in assessing how a genetic modification can be phenocopied by environmental conditions. In summary, these data demonstrate the use of metabolic profiling in conjunction with data-mining tools as a technique for the comprehensive characterization of a plant genotype.

Metabolite profiling for plant functional genomics.

Multiparallel analyses of mRNA and proteins are central to today's functional genomics initiatives. We describe here the use of metabolite profiling as a new tool for a comparative display of gene function. It has the potential not only to provide deeper insight into complex regulatory processes but also to determine phenotype directly. Using gas chromatography/mass spectrometry (GC/MS), we automatically quantified 326 distinct compounds from Arabidopsis thaliana leaf extracts. It was possible to assign a chemical structure to approximately half of these compounds. Comparison of four Arabidopsis genotypes (two homozygous ecotypes and a mutant of each ecotype) showed that each genotype possesses a distinct metabolic profile. Data mining tools such as principal component analysis enabled the assignment of "metabolic phenotypes" using these large data sets. The metabolic phenotypes of the two ecotypes were more divergent than were the metabolic phenotypes of the single-loci mutant and their parental ecotypes. These results demonstrate the use of metabolite profiling as a tool to significantly extend and enhance the power of existing functional genomics approaches.

Transgenic potato plants reveal the indispensable role of cystathionine beta-lyase in plant growth and development.

Methionine (Met) is an essential amino acid that is often unavailable at sufficient dietary levels. In order to better understand Met pathway regulation, a cDNA encoding cystathionine beta-lyase (CbL; EC 4.4.1.8) has been cloned from Solanum tuberosum. An antisense construct of this gene was used to generate transgenic potato plants with reduced CbL levels. Transgenic plants exhibiting leaf CbL activity levels of up to 50% below wild-type levels were obtained. Metabolite analysis revealed a reduction in Met levels in these CbL antisense plants, as well as remarkable increases in the pathway intermediates cystathionine, homoserine and cysteine. Unexpectedly, an increase in homocysteine was also observed. Levels of aspartate amino acid pathway intermediates (including aspartate, lysine and threonine) remained essentially unaffected. Neither transcript levels nor protein products of other pathway-relevant genes were altered significantly in these plants. CbL antisense plants exhibited an altered phenotype characterized by a bushy growth habit, small light-green leaves and small tubers. This phenotype could be alleviated upon Met supplementation, suggesting that low Met levels, rather than pathway intermediate accumulation, is responsible for the phenotypic effects of CbL transgene expression. These data unequivocally demonstrate the central role of CbL in Met biosynthesis, and, subsequently, in plant growth and development.

Comparative quantification and identification of phosphoproteins using stable isotope labeling and liquid chromatography/mass spectrometry.

A new liquid chromatography/mass spectrometry (LC/MS) method is described for relative quantification of phosphoproteins to simultaneously compare the phosphorylation status of proteins under two different conditions. Quantification was achieved by beta-elimination of phosphate from phospho-Ser/Thr followed by Micheal addition of ethanethiol and/or ethane-d(5)-thiol selectively at the vinyl moiety of dehydroalanine and dehydroamino-2-butyric acid. The method was evaluated using the model phosphoprotein alpha(S1)-casein, for which three phosphopeptides were found after tryptic digestion. Reproducibility of the relative quantification of seven independent replicates was found to be 11% SD. The dynamic range covered two orders of magnitude, and quantification was linear for mixtures of 0 to 100% alpha(S1)-casein and dephospho-alpha(S1)-casein (R(2) = 0.986). Additionally, the method allowed protein identification and determination of the phosphorylation sites via MS/MS fragmentation.

Antisense inhibition of plastidial phosphoglucomutase provides compelling evidence that potato tuber amyloplasts import carbon from the cytosol in the form of glucose-6-phosphate.

The aim of this work was to establish whether plastidial phosphoglucomutase is involved in the starch biosynthetic pathway of potato tubers and thereby to determine the form in which carbon is imported into the potato amyloplast. For this purpose, we cloned the plastidial isoform of potato PGM (StpPGM), and using an antisense approach generated transgenic potato plants that exhibited decreased expression of the StpPGM gene and contained significantly reduced total phosphoglucomutase activity. We confirmed that this loss in activity was due specifically to a reduction in plastidial PGM activity. Potato lines with decreased activities of plastidial PGM exhibited no major changes in either whole-plant or tuber morphology. However, tubers from these lines exhibited a dramatic (up to 40%) decrease in the accumulation of starch, and significant increases in the levels of sucrose and hexose phosphates. As tubers from these lines exhibited no changes in the maximal catalytic activities of other key enzymes of carbohydrate metabolism, we conclude that plastidial PGM forms part of the starch biosynthetic pathway of the potato tuber, and that glucose-6-phosphate is the major precursor taken up by amyloplasts in order to support starch synthesis.

Technical advance: simultaneous analysis of metabolites in potato tuber by gas chromatography-mass spectrometry.

A new method is presented in which gas chromatography coupled to mass spectrometry (GC-MS) allows the quantitative and qualitative detection of more than 150 compounds within a potato tuber, in a highly sensitive and specific manner. In contrast to other methods developed for metabolite analysis in plant systems, this method represents an unbiased and open approach that allows the detection of unexpected changes in metabolite levels. Although the method represents a compromise for a wide range of metabolites in terms of extraction, chemical modification and GC-MS analysis, for 25 metabolites analysed in detail the recoveries were found to be within the generally accepted range of 70-140%. Further, the reproducibility of the method was high: the error occurring in the analysis procedures was found to be less than 6% for 30 out of 33 compounds tested. Biological variability exceeded the systematic error of the analysis by a factor of up to 10. The method is also suited for upscaling, potentially allowing the simultaneous analysis of a large number of samples. As a first example this method has been applied to soil- and in vitro-grown tubers. Due to the simultaneous analysis of a wide range of metabolites it was immediately apparent that these systems differ significantly in their metabolism. Furthermore, the parallel insight into many pathways allows some conclusions to be drawn about the underlying physiological differences between both tuber systems. As a second example, transgenic lines modified in sucrose catabolism or starch synthesis were analysed. This example illustrates the power of an unbiased approach to detecting unexpected changes in transgenic lines.