Protease Inhibitors from Solanum chacoense Inhibit Pectobacterium Virulence by Reducing Bacterial Protease Activity and Motility.

Potato is a major staple crop, and necrotrophic bacterial pathogens such as Pectobacterium spp. are a major threat to global food security. Most lines of cultivated potato (Solanum tuberosum) are susceptible to Pectobacterium spp., but some lines of wild potato are resistant, including Solanum chacoense M6. Despite the discovery of resistance in wild potatoes, specific resistance genes are yet to be discovered. Crude protein extract from M6 had a global effect on Pectobacterium brasiliense Pb1692 (Pb1692) virulence phenotypes. Specifically, M6 protein extracts resulted in reduced Pectobacterium exo-protease activity and motility, induced cell elongation, and affected bacterial virulence and metabolic gene expression. These effects were not observed from protein extracts of susceptible potato S. tuberosum DM1. A proteomics approach identified protease inhibitors (PIs) as candidates for S. chacoense resistance, and genomic analysis showed higher abundance and diversity of PIs in M6 than in DM1. We cloned five PIs that are unique or had high abundance in M6 compared with DM1 and purified the proteins (g18987, g28531, g39249, g40384, g6571). Four of the PIs significantly reduced bacterial protease activity, with strongest effects from g28531 and g6571. Three PIs (g18987, g28531, g6571) inhibited disease when co-inoculated with Pectobacterium pathogens into potato tubers. Two PIs (g28531, g6571) also significantly reduced Pb1692 motility and are promising as resistance genes. These results show that S. chacoense PIs contribute to bacterial disease resistance by inhibiting exo-proteases, motility, and tuber maceration and by modulating cell morphology and metabolism. [Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Metabolomics reveals primary response of wheat (Triticum aestivum) to irrigation with oilfield produced water.

The reuse of oilfield produced water (PW) for agricultural irrigation has received increased attention for utility in drought-stricken regions. It was recently demonstrated that PW irrigation can affect physiological processes in food crops. However, metabolomic evaluations are important to further discern specific mechanisms of how PW may contribute as a plant-environmental stressor. Herein, the primary metabolic responses of wheat irrigated with PW and matching salinity controls were investigated. Non-targeted gas chromatography mass spectrometry (GC-MS) metabolomics was combined with multivariate analysis and revealed that PW irrigation altered the primary metabolic profiles of both wheat leaf and grain. Over 600 compounds (183 annotated metabolites) were detected that varied between controls (salinity control and tap water) and PW irrigated plants. While some of these changed metabolites are related to salinity stress, over half were found to be unique to PW. The primary metabolites exhibiting changes in abundance in leaf and grain tissues were amines/amino acids, organic acids, and saccharides. Metabolite pathway analysis revealed that amino acid metabolism, sugar metabolism, and nitrogen remobilization are all impacted by PW irrigation, independent of regular plant responses to salinity stress. These data, when combined with prior physiological studies, support a multi-faceted, physio-metabolic response of wheat to the unique stressor imposed by irrigation with PW.

Tafazzin deficiency impairs CoA-dependent oxidative metabolism in cardiac mitochondria.

Barth syndrome is a mitochondrial myopathy resulting from mutations in the tafazzin (TAZ) gene encoding a phospholipid transacylase required for cardiolipin remodeling. Cardiolipin is a phospholipid of the inner mitochondrial membrane essential for the function of numerous mitochondrial proteins and processes. However, it is unclear how tafazzin deficiency impacts cardiac mitochondrial metabolism. To address this question while avoiding confounding effects of cardiomyopathy on mitochondrial phenotype, we utilized Taz-shRNA knockdown (TazKD ) mice, which exhibit defective cardiolipin remodeling and respiratory supercomplex instability characteristic of human Barth syndrome but normal cardiac function into adulthood. Consistent with previous reports from other models, mitochondrial H2O2 emission and oxidative damage were greater in TazKD than in wild-type (WT) hearts, but there were no differences in oxidative phosphorylation coupling efficiency or membrane potential. Fatty acid and pyruvate oxidation capacities were 40-60% lower in TazKD mitochondria, but an up-regulation of glutamate oxidation supported respiration rates approximating those with pyruvate and palmitoylcarnitine in WT. Deficiencies in mitochondrial CoA and shifts in the cardiac acyl-CoA profile paralleled changes in fatty acid oxidation enzymes and acyl-CoA thioesterases, suggesting limitations of CoA availability or "trapping" in TazKD mitochondrial metabolism. Incubation of TazKD mitochondria with exogenous CoA partially rescued pyruvate and palmitoylcarnitine oxidation capacities, implicating dysregulation of CoA-dependent intermediary metabolism rather than respiratory chain defects in the bioenergetic impacts of tafazzin deficiency. These findings support links among cardiolipin abnormalities, respiratory supercomplex instability, and mitochondrial oxidant production and shed new light on the distinct metabolic consequences of tafazzin deficiency in the mammalian heart.

Metabolites from Wild Potato Inhibit Virulence Factors of the Soft Rot and Blackleg Pathogen Pectobacterium brasiliense.

Potato (Solanum tuberosum L.) is the primary vegetable crop consumed worldwide and is largely affected by bacterial pathogens that can cause soft rot and blackleg disease. Recently, resistance to these diseases has been identified in the wild potato S. chacoense, and the mechanism of resistance is unknown. Here, it was hypothesized that S. chacoense stems or tubers have unique chemistry that confers resistance to the pathogen Pectobacterium brasiliense through bactericidal, bacteriostatic, or antivirulence activity. Stem and tuber metabolite extracts were collected from S. chacoense and tested for effects on Pectobacterium bacterial multiplication rates, and activity and expression of known exoenzymes and virulence genes using S. tuberosum extracts as a comparative control. Comparatively, the S. chacoense extracts did not affect bacterial multiplication rate; however, they did reduce pectinase, cellulase, and protease activities. The chemical extracts were profiled using a bioassay-guided fractionation, and a nontargeted metabolomics comparison of S. chacoense and S. tuberosum stems and tubers was performed. The data showed that selected alkaloids, phenolic amines, phenols, amines, and peptides are integrative chemical sources of resistance against the bacteria.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Differential Stem Proteomics and Metabolomics Profiles for Four Wheat Cultivars in Response to the Insect Pest Wheat Stem Sawfly.

Common wheat (Triticum aestivum L.) is a global staple crop, and insect pests can impact grain yield. The wheat stem sawfly (Cephus cinctus, WSS) is a major wheat pest, and while partial resistance has been deployed by breeding for a solid-stem trait, this trait is affected by environment. Here, a proteomics and metabolomics study was performed on four wheat cultivars to characterize a molecular response to WSS infestation. The cultivars Hatcher (hollow-stem partially tolerant), Conan (semisolid-stem-resistant), and Denali and Reeder (hollow-stem-susceptible) were infested with WSS, and changes in stem proteins and metabolites were characterized using liquid chromatography-mass spectrometry. The proteome was characterized as 1830 proteins that included five major biological processes, including metabolic processes and response to stimuli, and the metabolome (1823 metabolites) spanned eight chemical superclasses, including alkaloids, benzenoids, and lipids. All four varieties had a molecular response to WSS following infestation. Hatcher had the most distinct changes, whereby 62 proteins and 29 metabolites varied in metabolic pathways involving enzymatic detoxification, proteinase inhibition, and antiherbivory compound production via benzoxazinoids, neolignans, and phenolics. Taken together, these data demonstrate variation in the wheat stem molecular response to WSS infestation and support breeding for molecular resistance in hollow-stem cultivars.

Non-Targeted Metabolomics Reveals Sorghum Rhizosphere-Associated Exudates are Influenced by the Belowground Interaction of Substrate and Sorghum Genotype.

Root exudation is an important plant process by which roots release small molecules into the rhizosphere that serve in overall plant functioning. Yet, there is a major gap in our knowledge in translating plant root exudation in artificial systems (i.e., hydroponics, sterile media) to crops, specifically for soils expected in field conditions. Sorghum (Sorghum bicolor L. Moench) root exudation was determined using both ultra-performance liquid chromatography and gas chromatography mass spectrometry-based non-targeted metabolomics to evaluate variation in exudate composition of two sorghum genotypes among three substrates (sand, clay, and soil). Above and belowground plant traits were measured to determine the interaction between sorghum genotype and belowground substrate. Plant growth and quantitative exudate composition were found to vary largely by substrate. Two types of changes to rhizosphere metabolites were observed: rhizosphere-enhanced metabolites (REMs) and rhizosphere-abated metabolites (RAMs). More REMs and RAMs were detected in sand and clay substrates compared to the soil substrate. This study demonstrates that belowground substrate influences the root exudate profile in sorghum, and that two sorghum genotypes exuded metabolites at different magnitudes. However, metabolite identification remains a major bottleneck in non-targeted metabolite profiling of the rhizosphere.

Obesity in mares promotes uterine inflammation and alters embryo lipid fingerprints and homeostasis.

Maternal body composition can be an important determinant for development of obesity and metabolic syndrome in adult offspring. Obesity-related outcomes in offspring may include epigenetic alterations; however, mechanisms of fetal programming remain to be fully elucidated. This study was conducted to determine the impact of maternal obesity in the absence of a high fat diet on equine endometrium and preimplantation embryos. Embryos were collected from normal and obese mares at 8 and 16 days and a uterine biopsy at 16 days (0 day = ovulation). With the exception of 8 day embryos, each sample was divided into two pieces. One piece was analyzed for gene expression markers related to carbohydrate metabolism, lipid homeostasis, inflammation, endoplasmic reticulum stress, oxidative stress, mitochondrial stress, and components of the insulin-like growth factor (IGF) system. The second piece was analyzed for lipid content using matrix-assisted laser desorption/ionization mass spectrometry. Obese mares had elevated concentrations of insulin, leptin, and total cholesterol, and they tended to have increased triglycerides and decreased insulin sensitivity. Embryos from obese mares had altered transcript abundance in genes for inflammation and lipid homeostasis, as well as endoplasmic reticulum, oxidative and mitochondrial stress and altered lipid fingerprints. Endometrium from obese mares had increased expression of inflammatory cytokines, lipid homeostasis regulation, mitochondrial stress, and the IGF2 system. This study demonstrates that increased adiposity in mares alters the uterine environment, transcript abundance of genes for cellular functions, and lipid profiles of embryos. These alterations could affect prenatal programming, with potential long-term effects in offspring.

Common bean varieties demonstrate differential physiological and metabolic responses to the pathogenic fungus Sclerotinia sclerotiorum.

Plant physiology and metabolism are important components of a plant response to microbial pathogens. Physiological resistance of common bean (Phaseolus vulgaris L.) to the fungal pathogen Sclerotinia sclerotiorum has been established, but the mechanisms of resistance are largely unknown. Here, the physiological and metabolic responses of bean varieties that differ in physiological resistance to S. sclerotiorum are investigated. Upon infection, the resistant bean variety A195 had a unique physiological response that included reduced photosynthesis and maintaining a higher leaf surface pH during infection. Leaf metabolomics was performed on healthy tissue adjacent to the necrotic lesion at 16, 24, and 48 hr post inoculation, and 144 metabolites were detected that varied between A195 and Sacramento following infection. The metabolites that varied in leaves included amines/amino acids, organic acids, phytoalexins, and ureides. The metabolic pathways associated with resistance included amine metabolism, uriede-based nitrogen remobilization, antioxidant production, and bean-specific phytoalexin production. A second experiment was conducted in stems of 13 bean genotypes with varying resistance. Stem resistance was associated with phytoalexin production, but unlike leaf metabolism, lipid changes were associated with susceptibility. Taken together, the data supports a multifaceted, physiometabolic response of common bean to S. sclerotiorum that mediates resistance.

GC-MS Metabolomics to Evaluate the Composition of Plant Cuticular Waxes for Four Triticum aestivum Cultivars.

Wheat (Triticum aestivum L.) is an important food crop, and biotic and abiotic stresses significantly impact grain yield. Wheat leaf and stem surface waxes are associated with traits of biological importance, including stress resistance. Past studies have characterized the composition of wheat cuticular waxes, however protocols can be relatively low-throughput and narrow in the range of metabolites detected. Here, gas chromatography-mass spectrometry (GC-MS) metabolomics methods were utilized to provide a comprehensive characterization of the chemical composition of cuticular waxes in wheat leaves and stems. Further, waxes from four wheat cultivars were assayed to evaluate the potential for GC-MS metabolomics to describe wax composition attributed to differences in wheat genotype. A total of 263 putative compounds were detected and included 58 wax compounds that can be classified (e.g., alkanes and fatty acids). Many of the detected wax metabolites have known associations to important biological functions. Principal component analysis and ANOVA were used to evaluate metabolite distribution, which was attributed to both tissue type (leaf, stem) and cultivar differences. Leaves contained more primary alcohols than stems such as 6-methylheptacosan-1-ol and octacosan-1-ol. The metabolite data were validated using scanning electron microscopy of epicuticular wax crystals which detected wax tubules and platelets. Conan was the only cultivar to display alcohol-associated platelet-shaped crystals on its abaxial leaf surface. Taken together, application of GC-MS metabolomics enabled the characterization of cuticular wax content in wheat tissues and provided relative quantitative comparisons among sample types, thus contributing to the understanding of wax composition associated with important phenotypic traits in a major crop.

Alternative acetate production pathways in Chlamydomonas reinhardtii during dark anoxia and the dominant role of chloroplasts in fermentative acetate production.

Chlamydomonas reinhardtii insertion mutants disrupted for genes encoding acetate kinases (EC 2.7.2.1) (ACK1 and ACK2) and a phosphate acetyltransferase (EC 2.3.1.8) (PAT2, but not PAT1) were isolated to characterize fermentative acetate production. ACK1 and PAT2 were localized to chloroplasts, while ACK2 and PAT1 were shown to be in mitochondria. Characterization of the mutants showed that PAT2 and ACK1 activity in chloroplasts plays a dominant role (relative to ACK2 and PAT1 in mitochondria) in producing acetate under dark, anoxic conditions and, surprisingly, also suggested that Chlamydomonas has other pathways that generate acetate in the absence of ACK activity. We identified a number of proteins associated with alternative pathways for acetate production that are encoded on the Chlamydomonas genome. Furthermore, we observed that only modest alterations in the accumulation of fermentative products occurred in the ack1, ack2, and ack1 ack2 mutants, which contrasts with the substantial metabolite alterations described in strains devoid of other key fermentation enzymes.

Employing Two-stage Derivatisation and GC-MS to Assay for Cathine and Related Stimulant Alkaloids across the Celastraceae.

INTRODUCTION: Catha edulis (qat, khat, mirra) is a woody plant species that is grown and consumed in East Africa and Yemen for its stimulant alkaloids cathinone, cathine and norephedrine. Two Celastraceae species, in addition to qat, have been noted for their stimulant properties in ethnobotanical literature. Recent phylogenetic reconstructions place four genera in a clade sister to Catha edulis, and these genera are primary candidates to search for cathine and related alkaloids. OBJECTIVE: Determine if cathine or related alkaloids are present in species of Celastraceae other than Catha edulis. METHODS: Leaf samples from 43 Celastraceae species were extracted in water followed by basification of the aqueous extract and partitioning with methyl-t-butyl ether to provide an alkaloid-enriched fraction. The extract was derivatised in a two-stage process and analysed using GC-MS for the presence of cathine. Related alkaloids and other metabolites in this alkaloid-enriched fraction were tentatively identified. RESULTS: Cathinone, cathine and norephedrine were not detected in any of the 43 Celastraceae species assayed other than Catha edulis. However, the phenylalanine- or tyrosine-derived alkaloid phenylethylamine was identified in five species. Nine species were found to be enriched for numerous sterol- and terpene-like compounds. CONCLUSION: These results indicate that cathine is unique to Catha edulis, and not the compound responsible for the stimulant properties reported in related Celastraceae species. Copyright © 2017 John Wiley & Sons, Ltd.

Effect of Obesity on the Preovulatory Follicle and Lipid Fingerprint of Equine Oocytes.

Obesity is associated with disrupted reproductive cycles in mares, but the impact of obesity on follicles and oocytes has received minimal attention. We investigated the impact of obesity on 1) expression of selected genes in follicle cells for carbohydrate metabolism, inflammatory cytokines, lipid homeostasis, endoplasmic reticulum stress, and mitochondrial function; 2) follicular fluid content of metabolic hormones and metabolites; and 3) lipid fingerprint of oocytes. Mares (9-13 yr) were classified as control (n = 8, normal weight, body condition score [BCS] 5.1, 10.4% body fat) or obese (n = 9, BCS 7.9, 16.2% body fat). Gene expression from granulosa cells (GC) and cumulus cells (CC) was evaluated by RT-PCR. Serum and follicular fluid were evaluated for insulin, leptin, adiponectin, and metabolite profiling. Oocyte lipid fingerprints were acquired using matrix-assisted laser desorption/ionization mass spectrometry. Several genes for lipid homeostasis, endoplasmic reticulum stress, and mitochondrial function were different between groups in GC and CC. Obese had (P < 0.05) or tended to have (0.05 < P < 0.1) lower insulin sensitivity and higher insulin and leptin in serum and follicular fluid. Many metabolites differed between control and obese in serum and/or follicular fluid and correlated with BCS and/or insulin sensitivity. Oocytes from control had greater concentrations of lipids consistent with phosphatidylcholines, phosphatidylethanolamines, and sphingomyelins, while lipids consistent with triglycerides tended to be higher in obese. These findings suggest that maternal obesity causes alterations in the follicle and oocyte; the extent to which these alterations impact the conceptus and offspring is still to be determined.

Inactivation of Phaeodactylum tricornutum urease gene using transcription activator-like effector nuclease-based targeted mutagenesis.

Diatoms are unicellular photosynthetic algae with promise for green production of fuels and other chemicals. Recent genome-editing techniques have greatly improved the potential of many eukaryotic genetic systems, including diatoms, to enable knowledge-based studies and bioengineering. Using a new technique, transcription activator-like effector nucleases (TALENs), the gene encoding the urease enzyme in the model diatom, Phaeodactylum tricornutum, was targeted for interruption. The knockout cassette was identified within the urease gene by PCR and Southern blot analyses of genomic DNA. The lack of urease protein was confirmed by Western blot analyses in mutant cell lines that were unable to grow on urea as the sole nitrogen source. Untargeted metabolomic analysis revealed a build-up of urea, arginine and ornithine in the urease knockout lines. All three intermediate metabolites are upstream of the urease reaction within the urea cycle, suggesting a disruption of the cycle despite urea production. Numerous high carbon metabolites were enriched in the mutant, implying a breakdown of cellular C and N repartitioning. The presented method improves the molecular toolkit for diatoms and clarifies the role of urease in the urea cycle.

Applying metabolomic analyses to the practice of embryology: physiology, development and assisted reproductive technology.

The advent of metabolomics technology and its application to small samples has allowed us to non-invasively monitor the metabolic activity of embryos in a complex culture environment. The aim of this study was to apply metabolomics technology to the analysis of individual embryos from several species during in vitro development to gain an insight into the metabolomics pathways used by embryos and their relationship with embryo quality. Alanine is produced by both in vivo- and in vitro-derived human, murine, bovine and porcine embryos. Glutamine is also produced by the embryos of these four species, but only those produced in vitro. Across species, blastocysts significantly consumed amino acids from the culture medium, whereas glucose was not significantly taken up. There are significant differences in the metabolic profile of in vivo- compared with in vitro-produced embryos at the blastocyst stage. For example, in vitro-produced murine embryos consume arginine, asparagine, glutamate and proline, whereas in vivo-produced embryos do not. Human embryos produce more alanine, glutamate and glutamine, and consume less pyruvate, at the blastocyst compared with cleavage stages. Glucose was consumed by human blastocysts, but not at a high enough level to reach significance. Consumption of tyrosine by cleavage stage human embryos is indicative of blastocyst development, although tyrosine consumption is not predictive of blastocyst quality. Similarly, although in vivo-produced murine blastocysts consumed less aspartate, lactate, taurine and tyrosine than those produced in vitro, consumption of these four amino acids by in vitro-derived embryos with high octamer-binding transcription factor 4 (Oct4) expression, indicative of high quality, did not differ from those with low Oct4 expression. Further application of metabolomic technologies to studies of the consumption and/or production of metabolites from individual embryos in a complete culture medium could transform our understanding of embryo physiology and improve our ability to produce developmentally competent embryos in vitro.

Application of nontargeted metabolite profiling to discover novel markers of quality traits in an advanced population of malting barley.

The process of breeding superior varieties for the agricultural industry is lengthy and expensive. Plant metabolites may act as markers of quality traits, potentially expediting the appraisal of experimental lines during breeding. Here, we evaluated the utility of metabolites as markers by assessing metabolic variation influenced by genetic and environmental factors in an advanced breeding setting and in relation to the phenotypic distribution of 20 quality traits. Nontargeted liquid chromatography-mass spectrometry metabolite profiling was performed on barley (Hordeum vulgare L.) grain and malt from 72 advanced malting barley lines grown at two distinct but climatically similar locations, with 2-row and 6-row barley as the main genetic factors. 27 420 molecular features were detected, and the metabolite and quality trait profiles were similarly influenced by genotype and environment; however, malt was more influenced by genotype compared with barley. An O2PLS model characterized molecular features and quality traits that covaried, and 1319 features associated with at least one of 20 quality traits. An indiscriminant MS/MS acquisition and novel data analysis method facilitated the identification of metabolites. The analysis described 216 primary and secondary metabolites that correlated with multiple quality traits and included amines, amino acids, alkaloids, polyphenolics and lipids. The mechanisms governing quality trait-metabolite associations were interpreted based on colocalization to genetic markers and their gene annotations. The results of this study support the hypothesis that metabolism and quality traits are co-influenced by relatively narrow genetic and environmental factors and illustrate the utility of grain metabolites as functional markers of quality traits.

Plant necrotrophic bacterial disease resistance phenotypes, QTL, and metabolites identified through integrated genetic mapping and metabolomics in Solanum species.

Most food crops are susceptible to necrotrophic bacteria that cause rotting and wilting diseases in fleshy organs and foods. All varieties of cultivated potato (Solanum tuberosum L.) are susceptible to diseases caused by Pectobacterium species, but resistance has been demonstrated in wild potato relatives including S. chacoense. Previous studies demonstrated that resistance is in part mediated by antivirulence activity of phytochemicals in stems and tubers. Little is known about the genetic basis of antivirulence traits, and the potential for inheritance and introgression into cultivated potato is unclear. Here, the metabolites and genetic loci associated with antivirulence traits in S. chacoense were elucidated by screening a sequenced S. tuberosum x S. chacoense recombinant inbred line (RIL) population for antivirulence traits of its metabolite extracts. Metabolite extracts from the RILs exhibited a quantitative distribution for two antivirulence traits that were positively correlated: quorum sensing inhibition and exo-protease inhibition, with some evidence of transgressive segregation, supporting the role of multiple loci and metabolites regulating these resistance-associated systems. Metabolomics was performed on the highly resistant and susceptible RILs that revealed 30 metabolites associated with resistance, including several alkaloids and terpenes. Specifically, several prenylated metabolites were more abundant in resistant RILs. We constructed a high-density linkage map with 795 SNPs mapped to 12 linkage groups, spanning a length of 1,507 cM and a density of 1 marker per 1.89 cM. Genetic mapping of the antivirulence and metabolite data identified five quantitative trait loci (QTLs) related to quorum sensing inhibition that explained 8-28% of the phenotypic variation and two QTLs for protease activity inhibition that explained 14-19% of the phenotypic variation. Several candidate genes including alkaloid, and secondary metabolite biosynthesis that are related to disease resistance were identified within these QTLs. Taken together, these data support that quorum sensing inhibition and exo-protease inhibition assays may serve as breeding targets to improve resistance to nectrotrophic bacterial pathogens in potato and other plants. The identified candidate genes and metabolites can be utilized in marker assisted selection and genomic selection to improve soft- rot and blackleg disease resistance.

"Retention projection" enables reliable use of shared gas chromatographic retention data across laboratories, instruments, and methods.

Gas chromatography/mass spectrometry (GC/MS) is a primary tool used to identify compounds in complex samples. Both mass spectra and GC retention times are matched to those of standards; however, it is often impractical to have standards on hand for every compound of interest, so we must rely on shared databases of MS data and GC retention information. Unfortunately, retention databases (e.g., linear retention index libraries) are experimentally restrictive, notoriously unreliable, and strongly instrument dependent, relegating GC retention information to a minor, often negligible role in compound identification despite its potential power. A new methodology called "retention projection" has great potential to overcome the limitations of shared chromatographic databases. In this work, we tested the reliability of the methodology in five independent laboratories. We found that, even when each lab ran nominally the same method, the methodology was 3-fold more accurate than retention indexing because it properly accounted for unintentional differences between the GC/MS systems. When the laboratories used different methods of their own choosing, retention projections were 4- to 165-fold more accurate. More importantly, the distribution of error in the retention projections was predictable across different methods and laboratories, thus enabling automatic calculation of retention time tolerance windows. Tolerance windows at 99% confidence were generally narrower than those widely used even when physical standards are on hand to measure their retention. With its high accuracy and reliability, the new retention projection methodology makes GC retention a reliable, precise tool for compound identification, even when standards are not available to the user.

Metabolic engineering of Arabidopsis for butanetriol production using bacterial genes.

1,2,4-butanetriol (butanetriol) is a useful precursor for the synthesis of the energetic material butanetriol trinitrate and several pharmaceutical compounds. Bacterial synthesis of butanetriol from xylose or arabinose takes place in a pathway that requires four enzymes. To produce butanetriol in plants by expressing bacterial enzymes, we cloned native bacterial or codon optimized synthetic genes under different promoters into a binary vector and stably transformed Arabidopsis plants. Transgenic lines expressing introduced genes were analyzed for the production of butanetriol using gas chromatography coupled to mass spectrometry (GC-MS). Soil-grown transgenic plants expressing these genes produced up to 20 µg/g of butanetriol. To test if an exogenous supply of pentose sugar precursors would enhance the butanetriol level, transgenic plants were grown in a medium supplemented with either xylose or arabinose and the amount of butanetriol was quantified. Plants expressing synthetic genes in the arabinose pathway showed up to a forty-fold increase in butanetriol levels after arabinose was added to the medium. Transgenic plants expressing either bacterial or synthetic xylose pathways, or the arabinose pathway showed toxicity symptoms when xylose or arabinose was added to the medium, suggesting that a by-product in the pathway or butanetriol affected plant growth. Furthermore, the metabolite profile of plants expressing arabinose and xylose pathways was altered. Our results demonstrate that bacterial pathways that produce butanetriol can be engineered into plants to produce this chemical. This proof-of-concept study for phytoproduction of butanetriol paves the way to further manipulate metabolic pathways in plants to enhance the level of butanetriol production.

Mitigation of obesity-promoted diseases by Nigella sativa and thymoquinone.

Obesity is closely associated with increased incidence of cardiovascular diseases, cancer, insulin resistance, and immune dysfunction, and thus obesity-mitigation strategies should take into account these secondary pathologies in addition to promoting weight loss. Recent studies indicate that black cumin (Nigella sativa) has cardio-protective, anti-cancer, anti-diabetic, antioxidant, and immune-modulatory properties. While black cumin and/or its major bioactive constituent, thymoquinone have demonstrated bioactivity in a variety of disease models, the mechanisms of action are largely unknown. Given the growing interest in and the use of functional foods and nutraceuticals, as well as the increase in obesity and chronic diseases worldwide, further research into the therapeutic/preventive effects of black cumin may be beneficial.

Grower decision-making factors in adoption of specialty cultivars: A case study of potatoes in the San Luis Valley.

Potatoes are the most consumed vegetable worldwide and play an important role in the U.S. economy. Growers make critical decisions each year in choosing which cultivar to grow, based on factors such as yield, resilience to the growing environment, and utility in the food industry. Current research supports the finding that less-common specialty cultivars (SCs) have benefits for human health. However, growers have been slow to adopt SCs into mainstream operations. Here, we identify major factors in the decision-making process that determine whether a population of growers in the San Luis Valley, Colorado, a major potato-growing region, adopt SC potatoes. We used a combination of ethnographic techniques and quantitative methods to examine drivers of adoption. The data demonstrate grower perceptions within potato farming and the complexity of interacting factors in decision-making. An integration of the Theory of Planned Behavior, Rational Expectation Hypothesis, and Diffusion of Innovation models identifies economic and social factors that influence grower decision-making. Growers that were more aware of specialty cultivar innovation and associated consumer demand were more open to SCs adoption. Other influencing factors include a grower's experience selling a SC in the previous year and access to diverse markets. Based on these data, we developed a new model to explain grower decision-making processes in adopting SCs. The model demonstrates that one current barrier to adoption is access to buyers, including warehouses, retailers, and households. Taken together, this research demonstrates how rational expectations stem from economic outcomes, knowledge, and experience in the potato industry. These results are important in helping to consider opportunities for growers to access new, higher value markets, while also improving consumer access to nutritious cultivars.