Scientific workflows facilitate the automation of data analysis tasks by integrating various software and tools executed in a particular order. To enable transparency and reusability in workflows, it is essential to implement the FAIR principles. Here, we describe our experiences implementing the FAIR principles for metabolomics workflows using the Metabolome Annotation Workflow (MAW) as a case study. MAW is specified using the Common Workflow Language (CWL), allowing for the subsequent execution of the workflow on different workflow engines. MAW is registered using a CWL description on WorkflowHub. During the submission process on WorkflowHub, a CWL description is used for packaging MAW using the Workflow RO-Crate profile, which includes metadata in Bioschemas. Researchers can use this narrative discussion as a guideline to commence using FAIR practices for their bioinformatics or cheminformatics workflows while incorporating necessary amendments specific to their research area.

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Transient expression in Nicotiana benthamiana offers a robust platform for the rapid production of complex secondary metabolites. It has proven highly effective in helping identify genes associated with pathways responsible for synthesizing various valuable natural compounds. While this approach has seen considerable success, it has yet to be applied to uncovering genes involved in anthocyanin biosynthetic pathways. This is because only a single anthocyanin, delphinidin 3‐O‐rutinoside, can be produced in N. benthamiana by activation of anthocyanin biosynthesis using transcription factors. The production of other anthocyanins would necessitate the suppression of certain endogenous flavonoid biosynthesis genes while transiently expressing others. In this work, we present a series of tools for the reconstitution of anthocyanin biosynthetic pathways in N. benthamiana leaves. These tools include constructs for the expression or silencing of anthocyanin biosynthetic genes and a mutant N. benthamiana line generated using CRISPR. By infiltration of defined sets of constructs, the basic anthocyanins pelargonidin 3‐O‐glucoside, cyanidin 3‐O‐glucoside and delphinidin 3‐O‐glucoside could be obtained in high amounts in a few days. Additionally, co‐infiltration of supplementary pathway genes enabled the synthesis of more complex anthocyanins. These tools should be useful to identify genes involved in the biosynthesis of complex anthocyanins. They also make it possible to produce novel anthocyanins not found in nature. As an example, we reconstituted the pathway for biosynthesis of Arabidopsis anthocyanin A5, a cyanidin derivative and achieved the biosynthesis of the pelargonidin and delphinidin variants of A5, pelargonidin A5 and delphinidin A5.

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Dalbergia melanoxylon Guill. & Perr (Fabaceae) is widely utilized in the traditional medicine of East Africa, showing effects against a variety of ailments including microbial infections. Phytochemical investigation of the root bark led to the isolation of six previously undescribed prenylated isoflavanones together with eight known secondary metabolites comprising isoflavanoids, neoflavones and an alkyl hydroxylcinnamate. Structures were elucidated based on HR-ESI-MS, 1- and 2-D NMR and ECD spectra. The crude extract and the isolated compounds of D. melanoxylon were tested for their antibacterial, antifungal, anthelmintic and cytotoxic properties, applying established model organisms non-pathogenic to humans. The crude extract exhibited significant antibacterial activity against Gram-positive Bacillus subtilis (97% inhibition at 50 μg/mL) and antifungal activity against the phytopathogens Phytophthora infestans, Botrytis cinerea and Septoria tritici (96, 89 and 73% at 125 μg/mL, respectively). Among the pure compounds tested, kenusanone H and (3R)-tomentosanol B exhibited, in a panel of partially human pathogenic bacteria and fungi, promising antibacterial activity against Gram-positive bacteria including methicillin-resistant Staphylococcus aureus (MRSA) and Mycobacterium showing MIC values between 0.8 and 6.2 μg/mL. The observed biological effects support the traditional use of D. melanoxylon and warrant detailed investigations of its prenylated isoflavanones as antibacterial lead compounds.

Over the past decades, Colombia has suffered complex social problems related to illicit crops, including forced displacement, violence, and environmental damage, among other consequences for vulnerable populations. Considerable effort has been made in the regulation of illicit crops, predominantly Cannabis sativa, leading to advances such as the legalization of medical cannabis and its derivatives, the improvement of crops, and leaving an open window to the development of scientific knowledge to explore alternative uses. It is estimated that C. sativa can produce approximately 750 specialized secondary metabolites. Some of the most relevant due to their anticancer properties, besides cannabinoids, are monoterpenes, sesquiterpenoids, triterpenoids, essential oils, flavonoids, and phenolic compounds. However, despite the increase in scientific research on the subject, it is necessary to study the primary and secondary metabolism of the plant and to identify key pathways that explore its great metabolic potential. For this purpose, a genome-scale metabolic reconstruction of C. sativa is described and contextualized using LC-QTOF-MS metabolic data obtained from the leaf extract from plants grown in the region of Pesca-Boyaca, Colombia under greenhouse conditions at the Clever Leaves facility. A compartmentalized model with 2101 reactions and 1314 metabolites highlights pathways associated with fatty acid biosynthesis, steroids, and amino acids, along with the metabolism of purine, pyrimidine, glucose, starch, and sucrose. Key metabolites were identified through metabolomic data, such as neurine, cannabisativine, cannflavin A, palmitoleic acid, cannabinoids, geranylhydroquinone, and steroids. They were analyzed and integrated into the reconstruction, and their potential applications are discussed. Cytotoxicity assays revealed high anticancer activity against gastric adenocarcinoma (AGS), melanoma cells (A375), and lung carcinoma cells (A549), combined with negligible impact against healthy human skin cells.

In biological discovery and engineering research, there is a need to spatially and/or temporally regulate transgene expression. However, the limited availability of promoter sequences that are uniquely active in specific tissue-types and/or at specific times often precludes co-expression of >multiple transgenes in precisely controlled developmental contexts. Here, we developed a system for use in rice that comprises synthetic designer transcription activator-like effectors (dTALEs) and cognate synthetic TALE-activated promoters (STAPs). The system allows multiple transgenes to be expressed from different STAPs, with the spatial and temporal context determined by a single promoter that drives expression of the dTALE. We show that two different systems—dTALE1-STAP1 and dTALE2-STAP2—can activate STAP-driven reporter gene expression in stable transgenic rice lines, with transgene transcript levels dependent on both dTALE and STAP sequence identities. The relative strength of individual STAP sequences is consistent between dTALE1 and dTALE2 systems but differs between cell-types, requiring empirical evaluation in each case. dTALE expression leads to off-target activation of endogenous genes but the number of genes affected is substantially less than the number impacted by the somaclonal variation that occurs during the regeneration of transformed plants. With the potential to design fully orthogonal dTALEs for any genome of interest, the dTALE-STAP system thus provides a powerful approach to fine-tune the expression of multiple transgenes, and to simultaneously introduce different synthetic circuits into distinct developmental contexts.

Liquid chromatography-mass spectrometry (LC-MS)-based untargeted metabolomics experiments have become increasingly popular because of the wide range of metabolites that can be analyzed and the possibility to measure novel compounds. LC-MS instrumentation and analysis conditions can differ substantially among laboratories and experiments, thus resulting in non-standardized datasets demanding customized annotation workflows. We present an ecosystem of R packages, centered around the MetaboCoreUtils, MetaboAnnotation and CompoundDb packages that together provide a modular infrastructure for the annotation of untargeted metabolomics data. Initial annotation can be performed based on MS1 properties such as m/z and retention times, followed by an MS2-based annotation in which experimental fragment spectra are compared against a reference library. Such reference databases can be created and managed with the CompoundDb package. The ecosystem supports data from a variety of formats, including, but not limited to, MSP, MGF, mzML, mzXML, netCDF as well as MassBank text files and SQL databases. Through its highly customizable functionality, the presented infrastructure allows to build reproducible annotation workflows tailored for and adapted to most untargeted LC-MS-based datasets. All core functionality, which supports base R data types, is exported, also facilitating its re-use in other R packages. Finally, all packages are thoroughly unit-tested and documented and are available on GitHub and through Bioconductor.

Agriculture is by far the biggest water consumer on our planet, accounting for 70 percent of all freshwater withdrawals. Climate change and a growing world population increase pressure on agriculture to use water more efficiently (‘more crop per drop’). Water‐use efficiency (WUE) and drought tolerance of crops are complex traits that are determined by many physiological processes whose interplay is not well understood. Here we describe a combinatorial engineering approach to optimize signaling networks involved in the control of stress tolerance. Screening a large population of combinatorially transformed plant lines, we identified a combination of calcium‐dependent protein kinase genes that confers enhanced drought stress tolerance and improved growth under water‐limiting conditions. Targeted introduction of this gene combination into plants increased plant survival under drought and enhanced growth under water‐limited conditions. Our work provides an efficient strategy for engineering complex signaling networks to improve plant performance under adverse environmental conditions, which does not depend on prior understanding of network function.

Background: different Solanaceae and Erythroxylaceae species produce tropane alkaloids. These alkaloids are the starting material in the production of different pharmaceuticals. The commercial demand for tropane alkaloids is covered by extracting them from cultivated plants. Datura stramonium is cultivated under greenhouse conditions as a source of tropane alkaloids. Here we investigate the effect of different levels of water availability in the soil on the production of tropane alkaloids by D. stramonium. Methods: We tested four irrigation levels on the accumulation of tropane alkaloids. We analyzed the profile of tropane alkaloids using an untargeted liquid chromatography/mass spectrometry method. Results: Using a combination of informatics and manual interpretation of mass spectra, we generated several structure hypotheses for signals in D. stramonium extracts that we assign as putative tropane alkaloids. Quantitation of mass spectrometry signals for our structure hypotheses across different anatomical organs allowed us to identify patterns of tropane alkaloids associated with different levels of irrigation. Furthermore, we identified anatomic partitioning of tropane alkaloid isomers with pharmaceutical applications. Conclusions: Our results show that soil water availability is an effective method for maximizing the production of specific tropane alkaloids for industrial applications.