A universal metabolite repair enzyme removes a strong inhibitor of the TCA cycle.

A prevalent side-reaction of succinate dehydrogenase oxidizes malate to enol-oxaloacetate (OAA), a metabolically inactive form of OAA that is a strong inhibitor of succinate dehydrogenase. We purified from cow heart mitochondria an enzyme (OAT1) with OAA tautomerase (OAT) activity that converts enol-OAA to the physiological keto-OAA form, and determined that it belongs to the highly conserved and previously uncharacterized Fumarylacetoacetate_hydrolase_domain-containing protein family. From all three domains of life, heterologously expressed proteins were shown to have strong OAT activity, and ablating the OAT1 homolog caused significant growth defects. In Escherichia coli, expression of succinate dehydrogenase was necessary for OAT1-associated growth defects to occur, and ablating OAT1 caused a significant increase in acetate and other metabolites associated with anaerobic respiration. OAT1 increased the succinate dehydrogenase reaction rate by 35% in in vitro assays with physiological concentrations of both succinate and malate. Our results suggest that OAT1 is a universal metabolite repair enzyme that is required to maximize aerobic respiration efficiency by preventing succinate dehydrogenase inhibition.

Post-secretory synthesis of a natural analog of iron-gall ink in the black nectar of Melianthus spp.

The black nectar produced by Melianthus flowers is thought to serve as a visual attractant to bird pollinators, but the chemical identity and synthesis of the black pigment are unknown. A combination of analytical biochemistry, transcriptomics, proteomics, and enzyme assays was used to identify the pigment that gives Melianthus nectar its black color and how it is synthesized. Visual modeling of pollinators was also used to infer a potential function of the black coloration. High concentrations of ellagic acid and iron give the nectar its dark black color, which can be recapitulated through synthetic solutions containing only ellagic acid and iron(iii). The nectar also contains a peroxidase that oxidizes gallic acid to form ellagic acid. In vitro reactions containing the nectar peroxidase, gallic acid, hydrogen peroxide, and iron(iii) fully recreate the black color of the nectar. Visual modeling indicates that the black color is highly conspicuous to avian pollinators within the context of the flower. Melianthus nectar contains a natural analog of iron-gall ink, which humans have used since at least medieval times. This pigment is derived from an ellagic acid-Fe complex synthesized in the nectar and is likely involved in the attraction of passerine pollinators endemic to southern Africa.

Using targeted metabolomics to elucidate the indole auxin network in plants.

The plant hormone auxin plays important roles throughout the entire life span of a plant and facilitates its adaptation to a changing environment. Multiple metabolic pathways intersect to control the levels and flux through indole-3-acetic acid (IAA), the primary auxin in most plant species. Measurement of changes in these pathways represents an important objective to understanding core aspects of auxin signal regulation. Such studies have become approachable through the technologies encompassed by targeted metabolomics. By monitoring incorporation of stable isotopes from labeled precursors into proposed intermediates, it is possible to trace pathway utilization and characterize new biosynthetic routes to auxin. Chemical inhibitors that target specific steps or entire pathways related to auxin synthesis aid these techniques. Here we describe methods for obtaining stable isotope labeled pathway intermediates necessary for pathway analysis and quantification of compounds. We describe how to use isotope dilution with methods employing either gas chromatography or high performance liquid chromatography mass spectrometry techniques for sensitive analysis of IAA. Complete biosynthetic pathway analysis in seedlings using multiple stable isotope-labeled precursors and chemical inhibitors coupled with highly sensitive liquid chromatography-mass spectrometry methods are described that allow rapid measurement of isotopic flux into biochemical pools. These methods should prove to be useful to researchers studying aspects of the auxin metabolic network in vivo in a variety of plant tissues and during various environmental conditions.

Developmental plasticity in multimodal signals: light environment produces novel signalling phenotypes in a butterfly.

Developmental plasticity can alter the expression of sexual signals in novel environments and is therefore thought to play an important role in promoting divergence. Sexual signals, however, are often multimodal and mate choice multivariate. Hence, to understand how developmental plasticity can facilitate divergence, we must assess plasticity across signal components and its cumulative impact on signalling. Here, we examine how developmental plasticity influences different components of cabbage white butterfly Pieris rapae multimodal signals, its effects on their signalling phenotypes and its implications for divergence. To do this, we reared P. rapae caterpillars under two different light environments (low-light and high-light) to simulate conditions experienced by P. rapae colonizing a novel light habitat. We then examined plasticity in both visual (wing coloration) and olfactory (pheromone abundance) components of male sexual signals. We found light environments influenced expression of both visual and olfactory components and resulted in a trade-off between signal modalities. The 'low-light' phenotype had duller wing colours but higher abundance of the pheromone, indole, whereas the 'high-light' phenotype had comparatively brighter wings but lower abundance of indole. These results show that by simultaneously altering expression of different signal components, developmental plasticity can produce multiple signalling phenotypes, which may catalyse divergence.

Convergent evolution of a blood-red nectar pigment in vertebrate-pollinated flowers.

Nearly 90% of flowering plants depend on animals for reproduction. One of the main rewards plants offer to pollinators for visitation is nectar. Nesocodon mauritianus (Campanulaceae) produces a blood-red nectar that has been proposed to serve as a visual attractant for pollinator visitation. Here, we show that the nectar's red color is derived from a previously undescribed alkaloid termed nesocodin. The first nectar produced is acidic and pale yellow in color, but slowly becomes alkaline before taking on its characteristic red color. Three enzymes secreted into the nectar are either necessary or sufficient for pigment production, including a carbonic anhydrase that increases nectar pH, an aryl-alcohol oxidase that produces a pigment precursor, and a ferritin-like catalase that protects the pigment from degradation by hydrogen peroxide. Our findings demonstrate how these three enzymatic activities allow for the condensation of sinapaldehyde and proline to form a pigment with a stable imine bond. We subsequently verified that synthetic nesocodin is indeed attractive to Phelsuma geckos, the most likely pollinators of Nesocodon We also identify nesocodin in the red nectar of the distantly related and hummingbird-visited Jaltomata herrerae and provide molecular evidence for convergent evolution of this trait. This work cumulatively identifies a convergently evolved trait in two vertebrate-pollinated species, suggesting that the red pigment is selectively favored and that only a limited number of compounds are likely to underlie this type of adaptation.

Metabolic signatures of Arabidopsis thaliana abiotic stress responses elucidate patterns in stress priming, acclimation, and recovery.

Temperature, water, and light are three abiotic stress factors that have major influences on plant growth, development, and reproduction. Plants can be primed by a prior mild stress to enhance their resistance to future stress. We used an untargeted metabolomics approach to examine Arabidopsis thaliana 11-day-old seedling's abiotic stress responses including heat (with and without priming), cold (with and without priming), water-deficit and high-light before and after a 2-day-recovery period. Analysis of the physiological phenotypes showed that seedlings with stress treatment resulted in a reduction in fresh weight, hypocotyl and root length but remained viable. Several stress responsive metabolites were identified, confirmed with reference standards, quantified, and clustered. We identified shared and specific stress signatures for cold, heat, water-deficit, and high-light treatments. Central metabolism including amino acid metabolism, sugar metabolism, glycolysis, TCA cycle, GABA shunt, glutathione metabolism, purine metabolism, and urea cycle were found to undergo changes that are fundamentally different, although some shared commonalities in response to different treatments. Large increases in cysteine abundance and decreases in reduced glutathione were observed following multiple stress treatments highlighting the importance of oxidative stress as a general phenomenon in abiotic stress. Large fold increases in low-turnover amino acids and maltose demonstrate the critical role of protein and starch autolysis in early abiotic stress responses.

Enzymes as Parts in Need of Replacement - and How to Extend Their Working Life.

Enzymes catalyze reactions in vivo at different rates and each enzyme molecule has a lifetime limit before it is degraded and replaced to enable catalysis to continue. Considering these rates together as a unitless ratio of catalytic cycles until replacement (CCR) provides a new quantitative tool to assess the replacement schedule of and energy investment into enzymes as they relate to function. Here, we outline the challenges of determining CCRs and new approaches to overcome them and then assess the CCRs of selected enzymes in bacteria and plants to reveal a range of seven orders of magnitude for this ratio. Modifying CCRs in plants holds promise to lower cellular costs, to tailor enzymes for particular environments, and to breed enzyme improvements for crop productivity.

Genetic analysis of stilbenoid profiles in grapevine stems reveals a major mQTL hotspot on chromosome 18 associated with disease-resistance motifs.

Grapevine (Vitis spp.) contains a wealth of phytochemicals that have received considerable attention due to health-promoting properties and biological activities as phytoalexins. To date, the genetic basis of the quantitative variations for these potentially beneficial compounds has been limited. Here, metabolic quantitative trait locus (mQTL) mapping was conducted using grapevine stems of a segregating F2 population. Metabolic profiling of grapevine stems was performed using liquid chromatography-high-resolution mass spectrometry (LC-HRMS), resulting in the detection of 1317 ions/features. In total, 19 of these features matched with literature-reported stilbenoid masses and were genetically mapped using a 1449-SNP linkage map and R/qtl software, resulting in the identification of four mQTLs. Two large-effect mQTLs that corresponded to a stilbenoid dimer and a trimer were mapped on chromosome 18, accounting for phenotypic variances of 29.0% and 38.4%. Functional annotations of these large-effect mQTLs on the VitisNet network database revealed a major hotspot of disease-resistance motifs on chromosome 18. This 2.8-Mbp region contains 48 genes with R-gene motifs, including variants of TIR, NBS, and LRR, that might potentially confer resistance to powdery mildew, downy mildew, or other pathogens. The locus also encompasses genes associated with flavonoid and biosynthetic pathways that are likely involved in the production of secondary metabolites, including phytoalexins. In addition, haplotype dosage effects of the five mQTLs further characterized the genomic regions for differential production of stilbenoids that can be applied in resistance breeding through manipulation of stilbenoid production in planta.

Leaf Spray Mass Spectrometry: A Rapid Ambient Ionization Technique to Directly Assess Metabolites from Plant Tissues.

Plants produce thousands of small molecules that are diverse in their chemical properties. Mass spectrometry (MS) is a powerful technique for analyzing plant metabolites because it provides molecular weights with high sensitivity and specificity. Leaf spray MS is an ambient ionization technique where plant tissue is used for direct chemical analysis via electrospray, eliminating chromatography from the process. This approach to sampling metabolites allows for a wide range of chemical classes to be detected simultaneously from intact plant tissues, minimizing the amount of sample preparation needed. When used with a high-resolution, accurate mass MS, leaf spray MS facilitates the rapid detection of metabolites of interest. It is also possible to collect tandem mass fragmentation data with this technique to facilitate a compound identification. The combination of accurate mass measurements and fragmentation is beneficial in confirming compound identities. The leaf spray MS technique requires only minor modifications to a nanospray ionization source and is a useful tool to further expand the capabilities of a mass spectrometer. Here, fresh leaf tissue from Sceletium tortuosum (Aizoaceae), a traditional medicinal plant from South Africa, is analyzed; numerous mesembrine alkaloids are detected with leaf spray MS.

Metabolic Patterns in Spirodela polyrhiza Revealed by 15N Stable Isotope Labeling of Amino Acids in Photoautotrophic, Heterotrophic, and Mixotrophic Growth Conditions.

In this study we describe a [15N] stable isotopic labeling study of amino acids in Spirodela polyrhiza (common duckweed) grown under three different light and carbon input conditions which represent unique potential metabolic modes. Plants were grown with a light cycle, either with supplemental sucrose (mixotrophic) or without supplemental sucrose (photoautotrophic) and in the dark with supplemental sucrose (heterotrophic). Labeling patterns, pool sizes (both metabolically active and inactive), and kinetics/turnover rates were estimated for 17 of the proteinogenic amino acids. Estimation of these parameters followed several overall trends. First, most amino acids showed plateaus in labeling patterns of <100% [15N]-labeling, indicating the possibility of a large proportion of amino acids residing in metabolically inactive metabolite pools. Second, total pool sizes appear largest in the dark (heterotrophic) condition, whereas active pool sizes appeared to be largest in the light with sucrose (mixotrophic) growth condition. In contrast turnover measurements based on pool size were highest overall in the light with sucrose experiment, with the exception of leucine/isoleucine, lysine, and arginine, which all showed higher turnover in the dark. K-means clustering analysis also revealed more rapid turnover in the light treatments with many amino acids clustering in lower-turnover groups. Emerging insights from other research were also supported, such as the prevalence of alternate pathways for serine metabolism in non-photosynthetic cells. These data provide extensive novel information on amino acid pool size and kinetics in S. polyrhiza and can serve as groundwork for future metabolic studies.

High Enrichment [13 C]-Labeling of Plants Grown Hydroponically from Seed to Seed in a Controlled 13 C-Carbon Dioxide Atmosphere Enclosure.

In vivo isotopic labeling empowers proteomic and metabolomic analyses to resolve relationships between the molecular composition, environment, and phenotype of an organism. Carbon-13 is particularly useful for plant labeling as it can be introduced via 13 CO2 gas and readily assimilated into plant metabolic systems through natural carbon fixation. While short-term labeling experiments can be performed within a simple sealed enclosure, long-term growth in an isolated environment raises many challenges beyond nutrient availability and buildup of metabolic waste. Viable growth conditions must be maintained by means that do not compromise the integrity of the carbon-13 enrichment. To address these issues, an automated growth chamber equipped with countermeasures to neutralize stresses and ensure high isotopic enrichment throughout the life cycle of the plant has been developed. The following describes this growth chamber and its use in an example 130-day growth of ten soybean plants to full maturity, achieving 100% carbon-13 enrichment of new seed tissue. © 2018 by John Wiley & Sons, Inc.

Improve your Galaxy text life: The Query Tabular Tool.

Galaxy provides an accessible platform where multi-step data analysis workflows integrating disparate software can be run, even by researchers with limited programming expertise. Applications of such sophisticated workflows are many, including those which integrate software from different 'omic domains (e.g. genomics, proteomics, metabolomics). In these complex workflows, intermediate outputs are often generated as tabular text files, which must be transformed into customized formats which are compatible with the next software tools in the pipeline. Consequently, many text manipulation steps are added to an already complex workflow, overly complicating the process. In some cases, limitations to existing text manipulation are such that desired analyses can only be carried out using highly sophisticated processing steps beyond the reach of even advanced users and developers. For users with some SQL knowledge, these text operations could be combined into single, concise query on a relational database. As a solution, we have developed the Query Tabular Galaxy tool, which leverages a SQLite database generated from tabular input data. This database can be queried and manipulated to produce transformed and customized tabular outputs compatible with downstream processing steps. Regular expressions can also be utilized for even more sophisticated manipulations, such as find and replace and other filtering actions. Using several Galaxy-based multi-omic workflows as an example, we demonstrate how the Query Tabular tool dramatically streamlines and simplifies the creation of multi-step analyses, efficiently enabling complicated textual manipulations and processing. This tool should find broad utility for users of the Galaxy platform seeking to develop and use sophisticated workflows involving text manipulation on tabular outputs.

Van Krevelen diagram visualization of high resolution-mass spectrometry metabolomics data with OpenVanKrevelen.

INTRODUCTION: Van Krevelen (VK) diagrams provide a promising but uncommon solution to a number of challenges associated with the visualization of metabolomics data. VK diagrams are created by plotting H:C ratios against O:C ratios of the compounds in a chemical mixture. OBJECTIVES: The aim of this manuscript is to present an open-source software tool and reference map that we have developed to make VK diagrams for visualization of metabolomics data. METHODS: Software was created with a prompt-driven command line user interface and was written using Python 2.7. We empirically derived an accompanying map by plotting where compounds from seven biomolecule types fall within the VK plot space. RESULTS: We've created an easy to use, open source software tool named OpenVanKrevelen for making a range of VK diagrams that is available on GitHub: https://github.com/HegemanLab/VanKrevelenLocal . The empirical mapping approach has produced several improvements from previously published maps. CONCLUSIONS: OpenVanKrevelen provides the metabolomics community with access to a new tool for visualization of complex metabolomics datasets.

Direct detection of surface localized specialized metabolites from Glycyrrhiza lepidota (American licorice) by leaf spray mass spectrometry.

MAIN CONCLUSION: Leaf spray-MS minimizes tissue manipulation by effectively and quickly assessing in vivo specialized metabolites from intact plant tissue surfaces, including trichome metabolites. Intact leaves of Glycyrrhiza lepidota Pursh. (American licorice) were analyzed by direct electrospray leaf spray-MS, an ambient ionization technique. Comparison of metabolites detected by leaf spray-MS to those from LC-MS of bulk tissue and trichome enriched extracts showed dramatic differences. Leaf spray-MS results suggest that in specific situations this approach could complement traditional LC-MS analysis of bulk extracts. Leaf spray-MS as a metabolomics technique eliminates sample pretreatment and preparation allowing for rapid sampling in real time of living intact tissues. Specialized metabolites on the surface of tissues such as glandular trichomes metabolites are detected by leaf spray-MS.

An improved method for fast and selective separation of carotenoids by LC-MS.

Carotenoids are a large class of compounds that are biosynthesized by condensation of isoprene units in plants, fungi, bacteria, and some animals. They are characteristically highly conjugated through double bonds, which lead to many isomers as well susceptibility to oxidation and other chemical modifications. Carotenoids are important because of their potent antioxidant activity and are the pigments responsible for color in a wide variety of foods. Human consumption is correlated to many health benefits including prevention of cancer, cardiovascular disease, and age-related disease. Extreme hydrophobicity, poor stability, and low concentration in biological samples make these compounds difficult to analyze and difficult to develop analytical methods for aimed towards identification and quantification. Examples in the literature frequently report the use of exotic stationary phases, solvents, and additives, such as ethyl acetate, dichloromethane, and methyl tert-butyl ether that are incompatible with liquid chromatography mass spectrometry (LC-MS). In order to address these issues, we implemented the use of LC-MS friendly conditions using a low-hydrophobicity cyano-propyl column (Agilent Zorbax SB-CN). We successfully differentiated between isomeric carotenoids by optimizing two gradient methods and using a mixture of 11 standards and LC-MS in positive ionization mode. Three complex biological samples from strawberry leaf, chicken feed supplement, and the photosynthetic bacterium Chloroflexus aurantiacus were analyzed and several carotenoids were resolved in these diverse backgrounds. Our results show this methodology is a significant improvement over other alternatives for analyzing carotenoids because of its ease of use, rapid analysis time, high selectivity, and, most importantly, its compatibility with typical LC-MS conditions.

3-Acyl dihydroflavonols from poplar resins collected by honey bees are active against the bee pathogens Paenibacillus larvae and Ascosphaera apis.

Honey bees, Apis mellifera, collect antimicrobial plant resins from the environment and deposit them in their nests as propolis. This behavior is of practical concern to beekeepers since the presence of propolis in the hive has a variety of benefits, including the suppression of disease symptoms. To connect the benefits that bees derive from propolis with particular resinous plants, we determined the identity and botanical origin of propolis compounds active against bee pathogens using bioassay-guided fractionation against the bacterium Paenibacillus larvae, the causative agent of American foulbrood. Eleven dihydroflavonols were isolated from propolis collected in Fallon, NV, including pinobanksin-3-octanoate. This hitherto unknown derivative and five other 3-acyl-dihydroflavonols showed inhibitory activity against both P. larvae (IC50 = 17-68 µM) and Ascosphaera apis (IC50 = 8-23 µM), the fungal agent of chalkbrood. A structure-activity relationship between acyl group size and antimicrobial activity was found, with longer acyl groups increasing activity against P. larvae and shorter acyl groups increasing activity against A. apis. Finally, it was determined that the isolated 3-acyl-dihydroflavonols originated from Populus fremontii, and further analysis showed these compounds can also be found in other North American Populus spp.

Recent advances in stable isotope-enabled mass spectrometry-based plant metabolomics.

Methods employing isotope labeled compounds have been an important part of the bioanalytical canon for many decades. The past fifteen years have seen the development of many new approaches using stable (non-radioactive) isotopes as labels for high-throughput bioanalytical, 'omics-scale' measurements of metabolites (metabolomics) and proteins (proteomics). This review examines stable isotopic labeling approaches that have been developed for labeling whole intact plants, plant tissues, or crude extracts of plant materials with stable isotopes (mainly using 2H, 13C, 15N, 18O or 34S). The application of metabolome-scale labeling for improving metabolite annotation, metabolic pathway elucidation, and relative quantification in mass spectrometry-based metabolomics of plants is also reviewed.

Measuring relative utilization of aerobic glycolysis in breast cancer cells by positional isotopic discrimination.

The ability of cancer cells to produce lactate through aerobic glycolysis is a hallmark of cancer. In this study, we established a positional isotopic labeling and LC-MS-based method that can specifically measure the conversion of glucose to lactate in glycolysis. We show that the rate of aerobic glycolysis is closely correlated with glucose uptake and lactate production in breast cancer cells. We also found that the production of [3-(13) C]lactate is significantly elevated in metastatic breast cancer cells and in early stage metastatic mammary tumors in mice. Our findings may enable the development of a biomarker for the diagnosis of aggressive breast cancer.

Extraction, purification, methylation and GC-MS analysis of short-chain carboxylic acids for metabolic flux analysis.

Dynamic metabolic flux analysis requires efficient and effective methods for extraction, purification and analysis of a plethora of naturally-occurring compounds. One area of metabolism that would be highly informative to study using metabolic flux analysis is the tricarboxylic acid (TCA) cycle, which consists of short-chain carboxylic acids. Here, we describe a newly-developed method for extraction, purification, derivatization and analysis of short-chain carboxylic acids involved in the TCA cycle. The method consists of snap-freezing the plant material, followed by maceration and a 12-15h extraction at -80 °C. The extracts are then subject to reduction (to stabilize ß-keto acids), purified by strong anion exchange solid phase extraction and methylated with methanolic HCl. This method could also be readily adapted to quantify many other short-chain carboxylic acids.