Metabolism of a sea lamprey pesticide by fish liver enzymes part B: method development and application in quantification of TFM metabolites formed in vivo.

The sea lamprey (Petromyzon marinus) is a destructive invasive species in the Great Lakes. Since the 1960s, tons of the lampricide 3-trifluoromethyl-4-nitrophenol (TFM) has been applied to selected tributaries each year to eliminate or reduce sea lamprey larval populations. Therefore, the environmental impact of TFM needs to be evaluated. However, the metabolism of TFM and its mechanism of selective toxicity in sea lamprey is not yet fully understood. Based upon our previous report on the identification, synthesis, and characterization of TFM metabolites observed in liver incubates from sea lamprey and non-target fishes, we now provide a robust assay for quantifying TFM and its metabolites in fish liver tissue. This method is important for assessing bioaccumulation of TFM in the ecosystems. The compounds purified in our previous report were used to develop and validate a quantitative ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) assay for TFM and TFM metabolites formed in vivo. Several sample preparation techniques were compared, and a protein precipitation method was selected. The unavailability of stable isotopic internal standards was overcome by using a matrix matching method. After a thorough validation, this method was applied to determine the concentrations of TFM and its metabolites in fish liver tissues from animals exposed to TFM, and in the comparison between dead animals and survivors. Seven of eight expected metabolites were observed, some for the first time in vivo. Our results indicate that in vivo nitroreduction, glucuronidation, sulfation, and glutathione conjugation are involved in TFM metabolism in sea lamprey.

Polyunsaturated fatty acids influence differential biosynthesis of oxylipids and other lipid mediators during bovine coliform mastitis.

Coliform mastitis is a severe and sometimes fatal disease characterized by an unregulated inflammatory response. The initiation, progression, and resolution of inflammatory responses are regulated, in part, by potent oxylipid metabolites derived from polyunsaturated fatty acids. The purpose of this study was to characterize the biosynthesis and diversity of oxylipid metabolites during acute bovine coliform mastitis. Eleven cows diagnosed with naturally occurring acute systemic coliform mastitis and 13 healthy control cows, matched for lactation number and days in milk, were selected for comparison of oxylipid and free fatty acid concentrations in both milk and plasma. Oxylipids and free fatty acids were quantified using liquid chromatography-tandem mass spectrometry. All polyunsaturated fatty acids quantified in milk were elevated during coliform mastitis with linoleic acid being the most abundant. Oxylipids synthesized through the lipoxygenase and cytochrome P450 pathways accounted for the majority of the oxylipid biosynthesis. This study demonstrated a complex and diverse oxylipid network, most pronounced at the level of the mammary gland. Substrate availability, biosynthetic pathways, and degree of metabolism influence the biosynthesis of oxylipids during bovine coliform mastitis. Further studies are required to identify targets for novel interventions that modulate oxylipid biosynthesis during coliform mastitis to optimize inflammation.

Trading acyls and swapping sugars: metabolic innovations in Solanum trichomes.

Solanaceae (nightshade family) species synthesize a remarkable array of clade- and tissue-specific specialized metabolites. Protective acylsugars, one such class of structurally diverse metabolites, are produced by AcylSugar AcylTransferases from sugars and acyl-coenzyme A esters. Published research revealed trichome acylsugars composed of glucose and sucrose cores in species across the family. In addition, acylsugars were analyzed across a small fraction of the >1200 species in the phenotypically megadiverse Solanum genus, with a handful containing inositol and glycosylated inositol cores. The current study sampled several dozen species across subclades of the Solanum to get a more detailed view of acylsugar chemodiversity. In depth characterization of acylsugars from the Clade II species Solanum melongena (brinjal eggplant) led to the identification of eight unusual structures with inositol or inositol glycoside cores, and hydroxyacyl chains. Liquid chromatography-mass spectrometry analysis of 31 additional species in the Solanum genus revealed striking acylsugar diversity with some traits restricted to specific clades and species. Acylinositols and inositol-based acyldisaccharides were detected throughout much of the genus. In contrast, acylglucoses and acylsucroses were more restricted in distribution. Analysis of tissue-specific transcriptomes and interspecific acylsugar acetylation differences led to the identification of the S. melongena AcylSugar AcylTransferase 3-Like 1 (SmASAT3-L1; SMEL4.1_12g015780) enzyme. This enzyme is distinct from previously characterized acylsugar acetyltransferases, which are in the ASAT4 clade, and appears to be a functionally divergent ASAT3. This study provides a foundation for investigating the evolution and function of diverse Solanum acylsugar structures and harnessing this diversity in breeding and synthetic biology.

Mass spectrometry screening reveals widespread diversity in trichome specialized metabolites of tomato chromosomal substitution lines.

Glandular secreting trichomes of cultivated tomato (Solanum lycopersicum) and close relatives produce a variety of structurally diverse volatile and non-volatile specialized ('secondary') metabolites, including terpenes, flavonoids and acyl sugars. A genetic screen is described here to profile leaf trichome and surface metabolite extracts of nearly isogenic chromosomal substitution lines covering the tomato genome. These lines contain specific regions of the Solanum pennellii LA0716 genome in an otherwise 'wild-type' M82 tomato genetic background. Regions that have an impact on the total amount of extractable mono- and sesquiterpenes (IL2-2) or only sesquiterpenes (IL10-3) or specifically influence accumulation of the monoterpene alpha-thujene (IL1-3 and IL1-4) were identified using GC-MS. A rapid LC-TOF-MS method was developed and used to identify changes in non-volatile metabolites through non-targeted analysis. Metabolite profiles generated using this approach led to the discovery of introgression lines producing different acyl chain substitutions on acyl sugar metabolites (IL1-3/1-4 and IL8-1/8-1-1), as well as two regions that influence the quantity of acyl sugars (IL5-3 and IL11-3). Chromosomal region 1-1/1-1-3 was found to influence the types of glycoalkaloids that are detected in leaf surface extracts. These results show that direct chemical screening is a powerful way to characterize genetic diversity in trichome specialized metabolism.

Evolutionary routes to biochemical innovation revealed by integrative analysis of a plant-defense related specialized metabolic pathway.

The diversity of life on Earth is a result of continual innovations in molecular networks influencing morphology and physiology. Plant specialized metabolism produces hundreds of thousands of compounds, offering striking examples of these innovations. To understand how this novelty is generated, we investigated the evolution of the Solanaceae family-specific, trichome-localized acylsugar biosynthetic pathway using a combination of mass spectrometry, RNA-seq, enzyme assays, RNAi and phylogenomics in different non-model species. Our results reveal hundreds of acylsugars produced across the Solanaceae family and even within a single plant, built on simple sugar cores. The relatively short biosynthetic pathway experienced repeated cycles of innovation over the last 100 million years that include gene duplication and divergence, gene loss, evolution of substrate preference and promiscuity. This study provides mechanistic insights into the emergence of plant chemical novelty, and offers a template for investigating the ~300,000 non-model plant species that remain underexplored.

The menD and menE homologs code for 2-succinyl-6-hydroxyl-2,4-cyclohexadiene-1-carboxylate synthase and O-succinylbenzoic acid-CoA synthase in the phylloquinone biosynthetic pathway of Synechocystis sp. PCC 6803.

The genome of the cyanobacterium Synechocystis sp. PCC 6803 contains genes identified as menD and menE, homologs of Escherichia coli genes that code for 2-succinyl-6-hydroxyl-2,4-cyclohexadiene-1-carboxylate (SHCHC) synthase and O-succinylbenzoic acid-CoA ligase in the menaquinone biosynthetic pathway. In cyanobacteria, the product of this pathway is 2-methyl-3-phytyl-1,4-naphthoquinone (phylloquinone), a molecule used exclusively as an electron transfer cofactor in Photosystem (PS) I. The menD(-) and menE(-) strains were generated, and both were found to lack phylloquinone. Hence, no alternative pathways exist in cyanobacteria to produce O-succinylbenzoyl-CoA. Q-band EPR studies of photoaccumulated quinone anion radical and optical kinetic studies of the P700(+) [F(A)/F(B)](-) backreaction indicate that in the mutant strains, plastoquinone-9 functions as the electron transfer cofactor in the A(1) site of PS I. At a light intensity of 40 microE m(-2) s(-1), the menD(-) and menE(-) mutant strains grew photoautotrophically and photoheterotrophically, but with doubling times slower than the wild type. Both of which are sensitive to high light intensities. Low-temperature fluorescence studies show that in the menD(-) and menE(-) mutants, the ratio of PS I to PS II is reduced relative to the wild type. Whole-chain electron transfer rates in the menD(-) and menE(-) mutant cells are correspondingly higher on a chlorophyll basis. The slower growth rate and high-light sensitivity of the menD(-) and menE(-) mutants are therefore attributed to a lower content of PS I per cell.

Triacylglycerol profiling of microalgae Chlamydomonas reinhardtii and Nannochloropsis oceanica.

Triacylglycerols (TAGs) from microalgae can serve as feedstock for the production of biofuels. To gain a comprehensive understanding of TAG metabolism in algae through genetic and molecular approaches, and to improve algal biofuel production, efficient and quantitative phenotyping methods focusing on TAGs are required. Towards this end, a facile ultrahigh performance liquid chromatography-mass spectrometry protocol was developed for TAG profiling, achieving identification and quantification of intact TAG molecular species in two algae. TAG profiling was performed in Chlamydomonas reinhardtii and Nannochloropsis oceanica grown in nitrogen (N)-replete or N-depleted medium. For the quantification of algal TAGs and fatty acids, two sets of internal standards were developed by taking advantage of the presence of pheophytin and specific fatty acids in algal samples. Comparison of algal TAG levels was simplified by using these internal standards for TAG analysis, paving the way for high-throughput mutant screening.