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Key message Phosphoglycerate Dehydrogenase 1 of the phosphorylated pathway of serine biosynthesis, active in heterotrophic plastids, is required for the synthesis of serine to enable plant growth at high rates of indolic glucosinolate biosynthesis. Abstract Plants have evolved effective strategies to defend against various types of pathogens. The synthesis of a multitude of specialized metabolites represents one effective approach to keep plant attackers in check. The synthesis of those defense compounds is cost intensive and requires extensive interaction with primary metabolism. However, how primary metabolism is adjusted to fulfill the requirements of specialized metabolism is still not completely resolved. Here, we studied the role of the phosphorylated pathway of serine biosynthesis (PPSB) for the synthesis of glucosinolates, the main class of defensive compounds in the model plant Arabidopsis thaliana. We show that major genes of the PPSB are co-expressed with genes required for the synthesis of tryptophan, the unique precursor for the formation of indolic glucosinolates (IG). Transcriptional and metabolic characterization of loss-of-function and dominant mutants of ALTERED TRYPTOPHAN1-like transcription factors revealed demand driven activation of PPSB genes by major regulators of IG biosynthesis. Trans-activation of PPSB promoters by ATR1/MYB34 transcription factor in cultured root cells confirmed this finding. The content of IGs were significantly reduced in plants compromised in the PPSB and these plants showed higher sensitivity against treatment with 5-methyl-tryptophan, a characteristic behavior of mutants impaired in IG biosynthesis. We further found that serine produced by the PPSB is required to enable plant growth under conditions of high demand for IG. In addition, PPSB-deficient plants lack the growth promoting effect resulting from interaction with the beneficial root-colonizing fungus Colletotrichum tofieldiae.

Because it is the precursor for various essential cellular components, the amino acid serine is indispensable for every living organism. In plants, serine is synthesized by two major pathways: photorespiration and the phosphorylated pathway of serine biosynthesis (PPSB). However, the importance of these pathways in providing serine for plant development is not fully understood. In this study, we examine the relative contributions of photorespiration and PPSB to providing serine for growth and metabolism in the C3 model plant Arabidopsis thaliana. Our analyses of cell proliferation and elongation reveal that PPSB-derived serine is indispensable for plant growth and its loss cannot be compensated by photorespiratory serine biosynthesis. Using isotope labeling, we show that PPSB-deficiency impairs the synthesis of proteins and purine nucleotides in plants. Furthermore, deficiency in PPSB-mediated serine biosynthesis leads to a strong accumulation of metabolites related to nitrogen metabolism. This result corroborates 15N-isotope labeling in which we observed an increased enrichment in labeled amino acids in PPSB-deficient plants. Expression studies indicate that elevated ammonium uptake and higher glutamine synthetase/glutamine oxoglutarate aminotransferase (GS/GOGAT) activity causes this phenotype. Metabolic analyses further show that elevated nitrogen assimilation and reduced amino acid turnover into proteins and nucleotides are the most likely driving forces for changes in respiratory metabolism and amino acid catabolism in PPSB-deficient plants. Accordingly, we conclude that even though photorespiration generates high amounts of serine in plants, PPSB-derived serine is more important for plant growth and its deficiency triggers the induction of nitrogen assimilation, most likely as an amino acid starvation response.

During photosynthesis, electrons travel from light-excited chlorophyll molecules along the electron transport chain to the final electron acceptor nicotinamide adenine dinucleotide phosphate (NADP) to form NADPH, which fuels the Calvin–Benson–Bassham cycle (CBBC). To allow photosynthetic reactions to occur flawlessly, a constant resupply of the acceptor NADP is mandatory. Several known stromal mechanisms aid in balancing the redox poise, but none of them utilizes the structurally highly similar coenzyme NAD(H). Using Arabidopsis (Arabidopsis thaliana) as a C3-model, we describe a pathway that employs the stromal enzyme PHOSPHOGLYCERATE DEHYDROGENASE 3 (PGDH3). We showed that PGDH3 exerts high NAD(H)-specificity and is active in photosynthesizing chloroplasts. PGDH3 withdrew its substrate 3-PGA directly from the CBBC. As a result, electrons become diverted from NADPH via the CBBC into the separate NADH redox pool. pgdh3 loss-of-function mutants revealed an overreduced NADP(H) redox pool but a more oxidized plastid NAD(H) pool compared to wild-type plants. As a result, photosystem I acceptor side limitation increased in pgdh3. Furthermore, pgdh3 plants displayed delayed CBBC activation, changes in nonphotochemical quenching, and altered proton motive force partitioning. Our fluctuating light-stress phenotyping data showed progressing photosystem II damage in pgdh3 mutants, emphasizing the significance of PGDH3 for plant performance under natural light environments. In summary, this study reveals an NAD(H)-specific mechanism in the stroma that aids in balancing the chloroplast redox poise. Consequently, the stromal NAD(H) pool may provide a promising target to manipulate plant photosynthesis.

The evolution of C4 photosynthesis led to an increase in carbon assimilation rates and plant growth compared to C3 photosynthetic plants. This enhanced plant growth, in turn, affects the requirement for soil-derived mineral nutrients. However, mineral plant nutrition has scarcely been considered in connection with C4 photosynthesis. Sulfur is crucial for plant growth and development, and preliminary studies in the genus Flaveria suggested metabolic differences in sulfate assimilation along the C4 evolutionary trajectory. Here, we show that in controlled conditions, foliar accumulation of the reduced sulfur compounds Cys and glutathione (GSH) increased with progressing establishment of the C4 photosynthetic cycle in different Flaveria species. An enhanced demand for reduced sulfur in C4Flaveria species is reflected in high rates of [35S]sulfate incorporation into GSH upon sulfate deprivation and increased GSH turnover as a reaction to the inhibition of GSH synthesis. Expression analyses indicate that the γ-glutamyl cycle is crucial for the recycling of GSH in C4 species. Sulfate reduction and GSH synthesis seems to be preferentially localized in the roots of C4 species, which might be linked to its colocalization with the phosphorylated pathway of Ser biosynthesis. Interspecies grafting experiments of F. robusta (C3) and F. bidentis (C4) revealed that the root system primarily controls sulfate acquisition, GSH synthesis, and sulfate and metabolite allocation in C3 and C4 plants. This study thus shows that evolution of C4 photosynthesis resulted in a wide range of adaptations of sulfur metabolism and points out the need for broader studies on importance of mineral nutrition for C4 plants.

Although the plant Phosphorylated Pathway of l-Ser Biosynthesis (PPSB) is essential for embryo and pollen development, and for root growth, its metabolic implications have not been fully investigated. A transcriptomics analysis of Arabidopsis (Arabidopsis thaliana) PPSB-deficient mutants at night, when PPSB activity is thought to be more important, suggested interaction with the sulfate assimilation process. Because sulfate assimilation occurs mainly in the light, we also investigated it in PPSB-deficient lines in the day. Key genes in the sulfate starvation response, such as the adenosine 5′phosphosulfate reductase genes, along with sulfate transporters, especially those involved in sulfate translocation in the plant, were induced in the PPSB-deficient lines. However, sulfate content was not reduced in these lines as compared with wild-type plants; besides the glutathione (GSH) steady-state levels in roots of PPSB-deficient lines were even higher than in wild type. This suggested that PPSB deficiency perturbs the sulfate assimilation process between tissues/organs. Alteration of thiol distribution in leaves from different developmental stages, and between aerial parts and roots in plants with reduced PPSB activity, provided evidence supporting this idea. Diminished PPSB activity caused an enhanced flux of (35)S into thiol biosynthesis, especially in roots. GSH turnover also accelerated in the PPSB-deficient lines, supporting the notion that not only biosynthesis, but also transport and allocation, of thiols were perturbed in the PPSB mutants. Our results suggest that PPSB is required for sulfide assimilation in specific heterotrophic tissues and that a lack of PPSB activity perturbs sulfur homeostasis between photosynthetic and nonphotosynthetic tissues.

The xylulose 5-phosphate/phosphate translocator (XPT) represents the fourth functional member of the phosphate translocator (PT) family residing in the plastid inner envelope membrane. In contrast to the other three members, little is known on the physiological role of the XPT. Based on its major transport substrates (i.e., pentose phosphates) the XPT has been proposed to act as a link between the plastidial and extraplastidial branches of the oxidative pentose phosphate pathway (OPPP). As the XPT is also capable of transporting triose phosphates, it might as well support the triose phosphate PT (TPT) in exporting photoassimilates from the chloroplast in the light (‘day path of carbon’) and hence in supplying the whole plant with carbohydrates. Two independent knockout mutant alleles of the XPT (xpt-1 and xpt-2) lacked any specific phenotype, suggesting that the XPT function is redundant. However, double mutants generated from crossings of xpt-1 to different mutant alleles of the TPT (tpt-1 and tpt-2) were severely retarded in size, exhibited a high chlorophyll fluorescence phenotype, and impaired photosynthetic electron transport rates. In the double mutant the export of triose phosphates from the chloroplasts is completely blocked. Hence, precursors for sucrose biosynthesis derive entirely from starch turnover (‘night path of carbon’), which was accompanied by a marked accumulation of maltose as a starch breakdown product. Moreover, pentose phosphates produced by the extraplastidial branch of the OPPP also accumulated in the double mutants. Thus, an active XPT indeed retrieves excessive pentose phosphates from the extra-plastidial space and makes them available to the plastids. Further metabolic profiling revealed that phosphorylated intermediates remained largely unaffected, whereas fumarate and glycine contents were diminished in the double mutants. The assessment of C/N-ratios suggested co-limitations of C- and N-metabolism as possible cause for growth retardation of the double mutants. Feeding of sucrose partially rescued the growth and photosynthesis phenotypes of the double mutants. Immunoblots of thylakoid proteins, spectroscopic determinations of photosynthesis complexes, and chlorophyll a fluorescence emission spectra at 77 Kelvin could only partially explain constrains in photosynthesis observed in the double mutants. The data are discussed together with aspects of the OPPP and central carbon metabolism.

Photorespiration is an essential pathway in photosynthetic organisms and is particularly important to detoxify and recycle 2-phosphoglycolate (2-PG), a by-product of oxygenic photosynthesis. The enzymes that catalyze the reactions in the photorespiratory core cycle and closely associated pathways have been identified; however, open questions remain concerning the metabolic network in which photorespiration is embedded. The amino acid serine represents one of the major intermediates in the photorespiratory pathway and photorespiration is thought to be the major source of serine in plants. The restriction of photorespiration to autotrophic cells raises questions concerning the source of serine in heterotrophic tissues. Recently, the phosphorylated pathway of serine biosynthesis has been found to be extremely important for plant development and metabolism. In this protocol, we describe a detailed methodological workflow to analyze the generative and vegetative phenotypes of plants deficient in the phosphorylated pathway of serine biosynthesis, which together allow a better understanding of its function in plants.

Photorespiration is an essential pathway in photosynthetic organisms and is particularly important to detoxify and recycle 2-phosphoglycolate (2-PG), a by-product of oxygenic photosynthesis. The enzymes that catalyze the reactions in the photorespiratory core cycle and closely associated pathways have been identified; however, open questions remain concerning the metabolic network in which photorespiration is embedded. The amino acid serine represents one of the major intermediates in the photorespiratory pathway and photorespiration is thought to be the major source of serine in plants. The restriction of photorespiration to autotrophic cells raises questions concerning the source of serine in heterotrophic tissues. Recently, the phosphorylated pathway of serine biosynthesis has been found to be extremely important for plant development and metabolism. In this protocol, we describe a detailed methodological workflow to analyze the generative and vegetative phenotypes of plants deficient in the phosphorylated pathway of serine biosynthesis, which together allow a better understanding of its function in plants.

Subcellular sugar partitioning in plants is strongly regulated in response to developmental cues and changes in external conditions. Besides transitory starch, the vacuolar sugars represent a highly dynamic pool of instantly accessible metabolites that serve as energy source and osmoprotectant. Here, we present the molecular identification and functional characterization of the vacuolar glucose (Glc) exporter Arabidopsis (Arabidopsis thaliana) Early Responsive to Dehydration-Like6 (AtERDL6). We demonstrate tonoplast localization of AtERDL6 in plants. In Arabidopsis, AtERDL6 expression is induced in response to factors that activate vacuolar Glc pools, like darkness, heat stress, and wounding. On the other hand, AtERDL6 transcript levels drop during conditions that trigger Glc accumulation in the vacuole, like cold stress and external sugar supply. Accordingly, sugar analyses revealed that Aterdl6 mutants have elevated vacuolar Glc levels and that Glc flux across the tonoplast is impaired under stress conditions. Interestingly, overexpressor lines indicated a very similar function for the ERDL6 ortholog Integral Membrane Protein from sugar beet (Beta vulgaris). Aterdl6 mutant plants display increased sensitivity against external Glc, and mutant seeds exhibit a 10% increase in seed weight due to enhanced levels of seed sugars, proteins, and lipids. Our findings underline the importance of vacuolar Glc export during the regulation of cellular Glc homeostasis and the composition of seed reserves.

Defining metabolite abundance and resulting fractional isotope enrichments, within and between cellular compartments, still remain a major challenge in modern plant biochemistry. Optimized protocols for rapid isolation of mitochondria (e.g., silicone oil centrifugation or membrane filters) or visualization of metabolites/metabolic states (e.g., fluorescence resonance energy transfer (FRET) or redox-sensitive fluorescent markers (roGFP)) have significantly improved and expanded our knowledge regarding mitochondrial metabolism. However, the application of nonaqueous fractionation (NAQF) to separate and quantify metabolites across subcellular compartments remains popular as a nontargeted, validated approach towards studying metabolism, and provides a top-down overview of metabolite distribution across the majority of the subcellular compartments in a single preparation. Unfortunately, of all the organelles resolved using this method, the mitochondrion still remains the most poorly defined. Here, the development and suggested improvements to resolve the mitochondrial metabolome are described.

Amino acid levels in plants are regulated by a complex interplay of regulatory circuits at the level of enzyme activities and gene expression. Despite the diversity of precursors involved in amino acid biosynthesis as providing the carbon backbones, the amino groups and, for the amino acids methionine and cysteine, the sulfhydryl group and despite the involvement of amino acids as substrates in various downstream metabolic processes, the plant usually manages to provide relatively constant levels of all amino acids. Here we collate data on how amino acid homeostasis is shifted upon depletion of one of the major biosynthetic constituents, i.e., sulfur. Arabidopsis thaliana seedlings exposed to sulfate starvation respond with a set of adaptation processes to achieve a new balance of amino acid metabolism. First, metabolites containing reduced sulfur (cysteine, glutathione, S-adenosylmethionine) are reduced leading to a number of downstream effects. Second, the relative excess accumulation of N over S triggers processes to dump nitrogen in asparagine, glutamine and further N-rich compounds like ureides. Third, the depletion of glutathione affects the redox and stress response system of the glutathione-ascorbate cycle. Thus, biosynthesis of aromatic compounds is triggered to compensate for this loss, leading to an increased flux and accumulation of aromatic amino acids, especially tryptophan. Despite sulfate starvation, the homeostasis is kept, though shifted to a new state. This adaptation process keeps the plant viable even under an adverse nutritional status.

Defining metabolite abundance and resulting fractional isotope enrichments, within and between cellular compartments, still remain a major challenge in modern plant biochemistry. Optimized protocols for rapid isolation of mitochondria (e.g., silicone oil centrifugation or membrane filters) or visualization of metabolites/metabolic states (e.g., fluorescence resonance energy transfer (FRET) or redox-sensitive fluorescent markers (roGFP)) have significantly improved and expanded our knowledge regarding mitochondrial metabolism. However, the application of nonaqueous fractionation (NAQF) to separate and quantify metabolites across subcellular compartments remains popular as a nontargeted, validated approach towards studying metabolism, and provides a top-down overview of metabolite distribution across the majority of the subcellular compartments in a single preparation. Unfortunately, of all the organelles resolved using this method, the mitochondrion still remains the most poorly defined. Here, the development and suggested improvements to resolve the mitochondrial metabolome are described.

In Ajuga reptans, raffinose oligosaccharides accumulated during winter. Stachyose, verbascose, and higher RFO oligomers were exclusively found in the vacuole whereas one-fourth of raffinose was localized in the stroma. The evergreen labiate Ajuga reptans L. can grow at low temperature. The carbohydrate metabolism changes during the cold phase, e.g., raffinose family oligosaccharides (RFOs) accumulate. Additionally, A. reptans translocates RFOs in the phloem. In the present study, subcellular concentrations of metabolites were studied in summer and winter leaves of A. reptans to gain further insight into regulatory instances involved in the cold acclimation process and into the function of RFOs. Subcellular metabolite concentrations were determined by non-aqueous fractionation. Volumes of the subcellular compartments of summer and winter leaves were analyzed by morphometric measurements. The metabolite content varied strongly between summer and winter leaves. Soluble metabolites increased up to tenfold during winter whereas the starch content was decreased. In winter leaves, the subcellular distribution showed a shift of carbohydrates from cytoplasm to vacuole and chloroplast. Despite this, the metabolite concentration was higher in all compartments in winter leaves compared to summer leaves because of the much higher total metabolite content in winter leaves. The different oligosaccharides did show different compartmentations. Stachyose, verbascose, and higher RFO oligomers were almost exclusively found in the vacuole whereas one-fourth of raffinose was localized in the stroma. Apparently, the subcellular distribution of the RFOs differs because they fulfill different functions in plant metabolism during winter. Raffinose might function in protecting chloroplast membranes during freezing, whereas higher RFO oligomers may exert protective effects on vacuolar membranes. In addition, the high content of RFOs in winter leaves may also result from reduced consumption of assimilates.

Amino acids serve as constituents of proteins, precursors for anabolism, and, in some cases, as signaling molecules in mammalians and plants. This review is focused on new insights, or speculations, on signaling functions of serine, γ-aminobutyric acid (GABA) and phenylalanine-derived phenylpropanoids. Serine acts as signal in brain tissue and mammalian cancer cells. In plants, de novo serine biosynthesis is also highly active in fast growing tissues such as meristems, suggesting a similar role of serine as in mammalians. GABA functions as inhibitory neurotransmitter in the brain. In plants, GABA is also abundant and seems to be involved in sexual reproduction, cell elongation, patterning and cell identity. The aromatic amino acids phenylalanine, tyrosine, and tryptophan are precursors for the production of secondary plant products. Besides their pharmaceutical value, lignans, neolignans and hydroxycinnamic acid amides (HCAA) deriving from phenylpropanoid metabolism and, in the case of HCAA, also from arginine have been shown to fulfill signaling functions or are involved in the response to biotic and abiotic stress. Although some basics on phenylpropanoid-derived signaling have been described, little is known on recognition- or signal transduction mechanisms. In general, mutant- and transgenic approaches will be helpful to elucidate the mechanistic basis of metabolite signaling.

Phosphoenolpyruvate (PEP) serves not only as a high energy carbon compound in glycolysis, but it acts also as precursor for plastidial anabolic sequences like the shikimate pathway, which produces aromatic amino acids (AAA) and subsequently secondary plant products. After conversion to pyruvate, PEP can also enter de novo fatty acid biosynthesis, the synthesis of branched-chain amino acids, and the non-mevalonate way of isoprenoid production. As PEP cannot be generated by glycolysis in chloroplasts and a variety of non-green plastids, it has to be imported from the cytosol by a phosphate translocator (PT) specific for PEP (PPT). A loss of function of PPT1 in Arabidopsis thaliana results in the chlorophyll a/b binding protein underexpressed1 (cue1) mutant, which is characterised by reticulate leaves and stunted roots. Here we dissect the shoot- and root phenotypes, and also address the question whether or not long distance signalling by metabolites is involved in the perturbed mesophyll development of cue1. Reverse grafting experiments showed that the shoot- and root phenotypes develop independently from each other, ruling out long distance metabolite signalling. The leaf phenotype could be transiently modified even in mature leaves, e.g. by an inducible PPT1RNAi approach or by feeding aromatic amino acids (AAA), the cytokinin trans-zeatin (tZ), or the putative signalling molecule dehydrodiconiferyl alcohol glucoside (DCG). Hormones, such as auxins, abscisic acid, gibberellic acid, ethylene, methyl jasmonate, and salicylic acid did not rescue the cue1 leaf phenotype. The low cell density1 (lcd1) mutant shares the reticulate leaf-, but not the stunted root phenotype with cue1. It could neither be rescued by AAA nor by tZ. In contrast, tZ and AAA further inhibited root growth both in cue1 and wild-type plants. Based on our results, we propose a model that PPT1 acts as a net importer of PEP into chloroplast, but as an overflow valve and hence exporter in root plastids.

Previously published studies have shown d-glutamate to be the most potent natural inhibitor of glutathione synthesis known, yet how d-glutamate is handled in humans is unknown. Therefore, we administered an oral d-glutamate load to four healthy volunteers and monitored the plasma d-glutamate concentration and excretion over a 3-h postload period. Compared with time controls, the plasma d-glutamate concentration increased 10-fold in the 1st h and then reached a plateau over the remaining time course. In contrast, plasma d-pyrrolidone carboxylic acid increased progressively throughout the 3-h time course to a level 10-fold higher than the d-glutamate plasma concentration. Excretion of d-glutamate progressively increased despite a constant filtered d-glutamate load rising from only 5 to 95% of the filtered amount. Excretion ofd-pyrrolidone carboxylic acid increased with the rise in filtered load without significant reabsorption. The amount ofd-pyrrolidone carboxylic acid excreted over the 3-h time course was 10 times the amount excreted as d-glutamate and accounted for almost 20% of the administered d-glutamate. These findings indicate that plasma d-glutamate concentration is tightly regulated through two mechanisms: 1) the transport into cells and metabolic conversion tod-pyrrolidone carboxylic acid and excretion, and 2) the enhancement of d-glutamate clearance by the kidneys.

Background: In patients with symptomatic carotid artery stenosis, high-intensity transient signals detected by transcranial Doppler (TCD) have been related to particulate microemboli originating at the stenotic lesion. The occurrence of these microembolic events within the Doppler spectrum should be influenced by antithrombotic agents of proven efficacy in these patients mainly by reducing cerebral embolism. Methods: Seventy-four of 192 consecutive patients with symptomatic arterial stenosis in the anterior circulation and clinical symptoms within the last 30 days underwent 1-hour bilateral TCD monitoring. Patients were selected, if they presented temporal bone windows enabling transcranial insonation, revealed normal Doppler CO2 test excluding hemodynamic impairment, had not received antithrombotic therapy other than acetylsalicylic acid (ASA) before sonographic examination, and gave informed consent to 1-hour monitoring which could be performed immediately on admission/presentation of the patient at the Department of Neurology. Results: Microembolic events were detected in 38 patients (51%). The proportion of patients with events among 26 patients without antithrombotic medication was 73% as compared with 40% in 48 patients receiving ASA at the time of TCD monitoring (p = 0.023). Multivariate analysis including time from ischemia to TCD, presence and start of ASA prevention, degree and localization of stenosis, and presence of a single or recurrent ischemia revealed that absence of an ASA prevention (odds ratio OR 7.1, 95% confidence interval CI 1.6–31.4, p = 0.010), recurrent ischemic events (OR 7.1, 95% CI 1.6–32.7, p = 0.011), and extracranial localization of the stenosis (OR 3.8, 95% CI 1.1–13.2, p = 0.038) were independent predictors for microembolic events. Conclusion: In patients with symptomatic arterial stenosis, the absence of an ASA medication is associated with the occurrence of TCD-detected microembolic events, suggesting a relation between these events and ASA-sensitive microemboli from the stenotic lesion.

Every biological organism relies for its proper function on interactions between a multitude of molecular entities like RNA, proteins, and metabolites. The comprehensive measurement and the analysis of all these entities would therefore provide the basis for our functional and mechanistic understanding of most biological processes. Next to their amount and identity, it is most crucial to also gain information about the subcellular distribution and the flux of the measured compounds between the cellular compartments. That is, we want to understand not only the individual functions of cellular components but also their functional implications within the whole organism. While the analysis of macromolecules like DNA, RNA, and proteins is quite established and robust, analytical techniques for small metabolites, which are prone to diffusion and degradation processes, provide a host of unsolved challenges. The major limitations here are the metabolite conversion and relocation processes. In this protocol we describe a methodological workflow which includes a nonaqueous fractionation method, a fractionated two-phase liquid/liquid extraction protocol, and a software package, which together allow extracting and analyzing starch, proteins, and especially polar and lipophilic metabolites from a single sample towards the estimation of their subcellular distributions.

Every biological organism relies for its proper function on interactions between a multitude of molecular entities like RNA, proteins, and metabolites. The comprehensive measurement and the analysis of all these entities would therefore provide the basis for our functional and mechanistic understanding of most biological processes. Next to their amount and identity, it is most crucial to also gain information about the subcellular distribution and the flux of the measured compounds between the cellular compartments. That is, we want to understand not only the individual functions of cellular components but also their functional implications within the whole organism. While the analysis of macromolecules like DNA, RNA, and proteins is quite established and robust, analytical techniques for small metabolites, which are prone to diffusion and degradation processes, provide a host of unsolved challenges. The major limitations here are the metabolite conversion and relocation processes. In this protocol we describe a methodological workflow which includes a nonaqueous fractionation method, a fractionated two-phase liquid/liquid extraction protocol, and a software package, which together allow extracting and analyzing starch, proteins, and especially polar and lipophilic metabolites from a single sample towards the estimation of their subcellular distributions.

In plants, two independent serine biosynthetic pathways, the photorespiratory and glycolytic phosphoserine (PS) pathways, have been postulated. Although the photorespiratory pathway is well characterized, little information is available on the function of the PS pathway in plants. Here, we present a detailed characterization of phosphoglycerate dehydrogenases (PGDHs) as components of the PS pathway in Arabidopsis thaliana. All PGDHs localize to plastids and possess similar kinetic properties, but they differ with respect to their sensitivity to serine feedback inhibition. Furthermore, analysis of pgdh1 and phosphoserine phosphatase mutants revealed an embryo-lethal phenotype and PGDH1-silenced lines were inhibited in growth. Metabolic analyses of PGDH1-silenced lines grown under ambient and high CO2 conditions indicate a direct link between PS biosynthesis and ammonium assimilation. In addition, we obtained several lines of evidence for an interconnection between PS and tryptophan biosynthesis, because the expression of PGDH1 and PHOSPHOSERINE AMINOTRANSFERASE1 is regulated by MYB51 and MYB34, two activators of tryptophan biosynthesis. Moreover, the concentration of tryptophan-derived glucosinolates and auxin were reduced in PGDH1-silenced plants. In essence, our results provide evidence for a vital function of PS biosynthesis for plant development and metabolism.

3'-Phosphoadenosine 5'-phosphosulfate (PAPS) is the high-energy sulfate donor for sulfation reactions. Plants produce some PAPS in the cytosol, but it is predominantly produced in plastids. Accordingly, PAPS has to be provided by plastids to serve as a substrate for sulfotransferase reactions in the cytosol and the Golgi apparatus. We present several lines of evidence that the recently described Arabidopsis thaliana thylakoid ADP/ATP carrier TAAC transports PAPS across the plastid envelope and thus fulfills an additional function of high physiological relevance. Transport studies using the recombinant protein revealed that it favors PAPS, 3'-phosphoadenosine 5'-phosphate, and ATP as substrates; thus, we named it PAPST1. The protein could be detected both in the plastid envelope membrane and in thylakoids, and it is present in plastids of autotrophic and heterotrophic tissues. TAAC/PAPST1 belongs to the mitochondrial carrier family in contrast with the known animal PAPS transporters, which are members of the nucleotide-sugar transporter family. The expression of the PAPST1 gene is regulated by the same MYB transcription factors also regulating the biosynthesis of sulfated secondary metabolites, glucosinolates. Molecular and physiological analyses of papst1 mutant plants indicate that PAPST1 is involved in several aspects of sulfur metabolism, including the biosynthesis of thiols, glucosinolates, and phytosulfokines.

With the development of high throughput metabolic technologies, a plethora of primary and secondary compounds have been detected in the plant cell. However, there are still major gaps in our understanding of the plant metabolome. This is especially true with regards to the compartmental localization of these identified metabolites.Non-aqueous fractionation (NAF) is a powerful technique for the determination of subcellular metabolite distributions in eukaryotic cells, and it has become the method of choice to analyze the distribution of a large number of metabolites concurrently. However, the NAF technique produces a continuous gradient of metabolite distributions, not discrete assignments. Resolution of these distributions requires computational analyses based on marker metabolites to resolve compartmental localizations.In this article we focus on expanding the computational analysis of data derived from non-aqueous fractionation. Along with an experimental workflow, we describe the critical steps in NAF experiments and how computational approaches can aid in assessing the quality and robustness of the derived data. For this, we have developed and provide a new version (v1.2) of the BestFit command line tool for calculation and evaluation of subcellular metabolite distributions. Furthermore, using both simulated and experimental data we show the influence on estimated subcellular distributions by modulating important parameters, such as the number of fractions taken or which marker metabolite is selected. Finally, we discuss caveats and benefits of non-aqueous fractionation analysis in the context of the compartmentalized metabolome.

Sulfur plays a pivotal role in the cellular metabolism of many organisms. In plants, the uptake and assimilation of sulfate is strongly regulated at the transcriptional level. Regulatory factors are the demand of reduced sulfur in organic or non-organic form and the level of O-acetylserine (OAS), the carbon precursor for cysteine biosynthesis. In plants, cysteine is synthesized by action of the cysteine-synthase complex (CSC) containing serine acetyltransferase (SAT) and O-acetylserine-(thiol)-lyase (OASTL). Both enzymes are located in plastids, mitochondria and the cytosol. The function of the compartmentation of the CSC to regulate sulfate uptake and assimilation is still not clearly resolved. To address this question, we analyzed Arabidopsis thaliana mutants for the plastidic and cytosolic SAT isoenzymes under sulfur starvation conditions. In addition, subcellular metabolite analysis by non-aqueous fractionation revealed distinct changes in subcellular metabolite distribution upon short-term sulfur starvation. Metabolite and transcript analyses of SERAT1.1 and SERAT2.1 mutants [previously analyzed in Krueger et al. (Plant Cell Environ 32:349-367, 2009)] grown under sulfur starvation conditions indicate that both isoenzymes do not contribute directly to the transcriptional regulation of genes involved in sulfate uptake and assimilation. Here, we summarize the current knowledge about the regulation of cysteine biosynthesis and the contribution of the different compartments to this metabolic process. We relate hypotheses and views of the regulation of cysteine biosynthesis with our results of applying sulfur starvation to mutants impaired in compartment-specific cysteine biosynthetic enzymes.

In plants, the enzymes for cysteine synthesis serine acetyltransferase (SAT) and O-acetylserine-(thiol)-lyase (OASTL) are present in the cytosol, plastids and mitochondria. However, it is still not clearly resolved to what extent the different compartments are involved in cysteine biosynthesis and how compartmentation influences the regulation of this biosynthetic pathway. To address these questions, we analysed Arabidopsis thaliana T-DNA insertion mutants for cytosolic and plastidic SAT isoforms. In addition, the subcellular distribution of enzyme activities and metabolite concentrations implicated in cysteine and glutathione biosynthesis were revealed by non-aqueous fractionation (NAF). We demonstrate that cytosolic SERAT1.1 and plastidic SERAT2.1 do not contribute to cysteine biosynthesis to a major extent, but may function to overcome transport limitations of O-acetylserine (OAS) from mitochondria. Substantiated by predominantly cytosolic cysteine pools, considerable amounts of sulphide and presence of OAS in the cytosol, our results suggest that the cytosol is the principal site for cysteine biosynthesis. Subcellular metabolite analysis further indicated efficient transport of cysteine, gamma-glutamylcysteine and glutathione between the compartments. With respect to regulation of cysteine biosynthesis, estimation of subcellular OAS and sulphide concentrations established that OAS is limiting for cysteine biosynthesis and that SAT is mainly present bound in the cysteine-synthase complex.

Bioinformatics, 21 (8), ISSN:1367-4803, ISSN:1460-2059

Background and Purpose— We aimed to investigate whether the time period of transcranial Doppler monitoring for embolic signals can be reduced without loss of clinical yield compared with routinely performed 1-hour monitoring. Methods— Investigations on the basis of a post hoc analysis of a previously published cohort of 86 patients (55 men, 31 women; mean age 60.6 years) with a nondisabling arterioembolic ischemic event in the anterior circulation within the last 30 days (mean 7.3) and an ipsilateral medium-grade or high-grade stenosis of the carotid or middle cerebral artery. Patients underwent 1-hour monitoring for embolic signals and were followed up prospectively for 6 weeks to evaluate the relationship between embolic signals and risk of an early ischemic recurrence. Risk was also calculated after fictitious reduction of the monitoring period from 60 minutes to 50, 40, 30, 20, and 10 minutes, respectively, and compared with the results obtained from the 1-hour period. Results— The number of patients positive for embolic signals decreased with the decreasing monitoring period. By this, the odds ratio of embolic signals for an early ischemic recurrence “decreased” from 40 (derived from the 1-hour monitoring) to 10 when the monitoring lasted ≤30 minutes. The relationship between the rate of embolic signals per hour and risk of a recurrent stroke is described by an S-shaped curve. As a consequence, risk estimated from reduced monitoring periods can differ considerably from that derived from the 1-hour monitoring if the signal frequency lies within a medium range (eg, between 3 and 15 signals in 30 minutes). Conclusions— The time period of monitoring for embolic signals may be reduced without loss of clinical relevant information when signal frequency is low or already high during the reduced monitoring period, but it should be prolonged to maximally an hour at signal numbers within a medium range. However, our results need to be externally validated on an independent cohort of patients or confirmed by a prospective study before this modification can be recommended in general.

The expression of platelet-derived growth factor-A (PDGF-A) mRNA was examined in the cotyledons of normal human placentae and those from patients with pre-eclampsia. These patients exhibited pre-delivery blood pressure of 154 ± 4/99 ± 4 mmHg (mean ± SEM) and met the criteria established for pre-eclampsia. During labour they received MgS04 infusion for various time intervals (4–25 h). The PDGF-A message was quantitated to β-actin by the solution hybridization nuclease protection assay. Since the two groups differed in two parameters (pre-eclampsia and MgSO4 treatment), the direct comparison was not feasible. An analysis of covariance revealed a significant difference in the message between the pre-eclamptic and control groups (P

Background We have studied the impact of carbohydrate-starvation on the acclimation response to high light using Arabidopsis thaliana double mutants strongly impaired in the day- and night path of photoassimilate export from the chloroplast. A complete knock-out mutant of the triose phosphate/phosphate translocator (TPT; tpt-2 mutant) was crossed to mutants defective in (i) starch biosynthesis (adg1-1, pgm1 and pgi1-1; knock-outs of ADP-glucose pyrophosphorylase, plastidial phosphoglucomutase and phosphoglucose isomerase) or (ii) starch mobilization (sex1-3, knock-out of glucan water dikinase) as well as in (iii) maltose export from the chloroplast (mex1-2). Results All double mutants were viable and indistinguishable from the wild type when grown under low light conditions, but - except for sex1-3/tpt-2 - developed a high chlorophyll fluorescence (HCF) phenotype and growth retardation when grown in high light. Immunoblots of thylakoid proteins, Blue-Native gel electrophoresis and chlorophyll fluorescence emission analyses at 77 Kelvin with the adg1-1/tpt-2 double mutant revealed that HCF was linked to a specific decrease in plastome-encoded core proteins of both photosystems (with the exception of the PSII component cytochrome b559), whereas nuclear-encoded antennae (LHCs) accumulated normally, but were predominantly not attached to their photosystems. Uncoupled antennae are the major cause for HCF of dark-adapted plants. Feeding of sucrose or glucose to high light-grown adg1-1/tpt-2 plants rescued the HCF- and growth phenotypes. Elevated sugar levels induce the expression of the glucose-6-phosphate/phosphate translocator2 (GPT2), which in principle could compensate for the deficiency in the TPT. A triple mutant with an additional defect in GPT2 (adg1-1/tpt-2/gpt2-1) exhibited an identical rescue of the HCF- and growth phenotype in response to sugar feeding as the adg1-1/tpt-2 double mutant, indicating that this rescue is independent from the sugar-triggered induction of GPT2. Conclusions We propose that cytosolic carbohydrate availability modulates acclimation to high light in A. thaliana. It is conceivable that the strong relationship between the chloroplast and nucleus with respect to a co-ordinated expression of photosynthesis genes is modified in carbohydrate-starved plants. Hence carbohydrates may be considered as a novel component involved in chloroplast-to-nucleus retrograde signaling, an aspect that will be addressed in future studies.

Inhibition of Growth Factor-Modulated Vascular Smooth Muscle Cell Hyperplasia by Trapidil, Pentoxifylline and Magnesium Sulfate

Vascular Endothelial Growth Factor is Suppressed in the Plasma of Preeclamptic Patients † 776