Diatoms (Bacillariophyceae) are aquatic photosynthetic microalgae with an ecological role as primary producers in the aquatic food web. They account substantially for global carbon, nitrogen, and silicon cycling. Elucidating the chemical space of diatoms is crucial to understanding their physiology and ecology. To expand the known chemical space of a cosmopolitan marine diatom, Skeletonema marinoi, we performed High-Resolution Liquid Chromatography-Tandem Mass Spectrometry (LC-MS2) for untargeted metabolomics data acquisition. The spectral data from LC-MS2 was used as input for the Metabolome Annotation Workflow (MAW) to obtain putative annotations for all measured features. A suspect list of metabolites previously identified in the Skeletonema spp. was generated to verify the results. These known metabolites were then added to the putative candidate list from LC-MS2 data to represent an expanded catalog of 1970 metabolites estimated to be produced by S. marinoi. The most prevalent chemical superclasses, based on the ChemONT ontology in this expanded dataset, were organic acids and derivatives, organoheterocyclic compounds, lipids and lipid-like molecules, and organic oxygen compounds. The metabolic profile from this study can aid the bioprospecting of marine microalgae for medicine, biofuel production, agriculture, and environmental conservation. The proposed analysis can be applicable for assessing the chemical space of other microalgae, which can also provide molecular insights into the interaction between marine organisms and their role in the functioning of ecosystems.

Mapping the chemical space of compounds to chemical structures remains a challenge in metabolomics. Despite the advancements in untargeted liquid chromatography-mass spectrometry (LC–MS) to achieve a high-throughput profile of metabolites from complex biological resources, only a small fraction of these metabolites can be annotated with confidence. Many novel computational methods and tools have been developed to enable chemical structure annotation to known and unknown compounds such as in silico generated spectra and molecular networking. Here, we present an automated and reproducible Metabolome Annotation Workflow (MAW) for untargeted metabolomics data to further facilitate and automate the complex annotation by combining tandem mass spectrometry (MS2) input data pre-processing, spectral and compound database matching with computational classification, and in silico annotation. MAW takes the LC-MS2 spectra as input and generates a list of putative candidates from spectral and compound databases. The databases are integrated via the R package Spectra and the metabolite annotation tool SIRIUS as part of the R segment of the workflow (MAW-R). The final candidate selection is performed using the cheminformatics tool RDKit in the Python segment (MAW-Py). Furthermore, each feature is assigned a chemical structure and can be imported to a chemical structure similarity network. MAW is following the FAIR (Findable, Accessible, Interoperable, Reusable) principles and has been made available as the docker images, maw-r and maw-py. The source code and documentation are available on GitHub (https://github.com/zmahnoor14/MAW). The performance of MAW is evaluated on two case studies. MAW can improve candidate ranking by integrating spectral databases with annotation tools like SIRIUS which contributes to an efficient candidate selection procedure. The results from MAW are also reproducible and traceable, compliant with the FAIR guidelines. Taken together, MAW could greatly facilitate automated metabolite characterization in diverse fields such as clinical metabolomics and natural product discovery.

Jasmonates (JAs), including jasmonic acid (JA) and its biologically active derivative JA-Ile, are lipid-derived plant signaling molecules. They govern plant responses to stresses, such as wounding and insect herbivory. Wounding elicits a rapid increase of JA and JA-Ile levels as well as the expression of JAR1, coding for the enzyme involved in JA-Ile biosynthesis. Endogenous increase and application of JAs, such as MeJA, a JA methylester, result in increased defense levels, often accompanied by diminished growth. A JA-Ile biosynthesis inhibitor, jarin-1, was shown to exclusively inhibit the JA-conjugating enzyme JAR1 in Arabidopsis thaliana. To investigate whether jarin-1 does function similarly in other plants, we tested this in Medicago truncatula, Solanum lycopersicum, and Brassica nigra seedlings in a root growth inhibition assay. Application of jarin-1 alleviated the inhibition of root growth after MeJA application in M. truncatula seedlings, proving that jarin-1 is biologically active in M. truncatula. Jarin-1 did not show, however, a similar effect in S. lycopersicum and B. nigra seedlings treated with MeJA. Even JA-Ile levels were not affected by application of jarin-1 in wounded leaf disks from S. lycopersicum. Based on these results, we conclude that the effect of jarin-1 is highly species-specific. Researchers intending to use jarin-1 for studying the function of JAR1 or JA-Ile in their model plants, must test its functionality before use.

Ethylene (ET) controls many facets of plant growth and development under abiotic and biotic stresses. MtEIN2, as a critical element of the ET signaling pathway, is essential in biotic interactions. However, the role of MtEIN2 in responding to abiotic stress, such as combined nutrient deficiency, is less known. To assess the role of ethylene signaling in nutrient uptake, we manipulated nitrate (NO3−) and phosphate (Pi) availability for wild-type (WT) and the ethylene-insensitive (MtEIN2-defective) mutant, sickle, in Medicago truncatula. We measured leaf biomass and photosynthetic pigments in WT and sickle to identify conditions leading to different responses in both genotypes. Under combined NO3− and Pi deficiency, sickle plants had higher chlorophyll and carotenoid contents than WT plants. Under these conditions, nitrate content and gene expression levels of nitrate transporters were higher in the sickle mutant than in the WT. This led to the conclusion that MtEIN2 is associated with nitrate uptake and the content of photosynthetic pigments under combined Pi and NO3−deficiency in M. truncatula. We conclude that ethylene perception plays a critical role in regulating the nutrient status of plants.

Plant breeding and genetics play a major role in the adaptation of plants to meet human needs. The current requirement to make agriculture more sustainable can be partly met by a greater reliance on biological nitrogen fixation (BNF) by symbiotic diazotrophic microorganisms that provide crop plants with ammonium. Select accessions of the cereal crop sorghum (Sorghum bicolor (L.) Moench) form mucilage-producing aerial roots that harbor nitrogen-fixing bacteria. Breeding programs aimed at developing sorghum varieties that support diazotrophs will benefit from a detailed understanding of the genetic and environmental factors contributing to aerial root formation. A genome-wide association study (GWAS) of the sorghum minicore, a collection of 242 landraces, and 30 accessions from the sorghum association panel (SAP) was conducted in Florida and Wisconsin and under two fertilizer treatments to identify loci associated with the number of nodes with aerial roots and aerial root diameter. Sequence variation in genes encoding transcription factors that control phytohormone signaling and root system architecture showed significant associations with these traits. In addition, the location had a significant effect on the phenotypes. Concurrently, we developed F2 populations from crosses between bioenergy sorghums and a landrace that produced extensive aerial roots to evaluate the mode of inheritance of the loci identified by the GWAS. Furthermore, the mucilage collected from aerial roots contained polysaccharides rich in galactose, arabinose, and fucose, whose composition displayed minimal variation among 10 genotypes and two fertilizer treatments. These combined results support the development of sorghums with the ability to acquire nitrogen via BNF.

The metabolome is the biochemical basis of plant form and function, but we know little about its macroecological variation across the plant kingdom. Here, we used the plant functional trait concept to interpret leaf metabolome variation among 457 tropical and 339 temperate plant species. Distilling metabolite chemistry into five metabolic functional traits reveals that plants vary on two major axes of leaf metabolic specialization—a leaf chemical defense spectrum and an expression of leaf longevity. Axes are similar for tropical and temperate species, with many trait combinations being viable. However, metabolic traits vary orthogonally to life-history strategies described by widely used functional traits. The metabolome thus expands the functional trait concept by providing additional axes of metabolic specialization for examining plant form and function.

Macrocyclization of peptides is typically used to fix specific bioactive conformations and improve their pharmacological properties. Recently, macrobicyclic peptides have received special attention owing to their capacity to mimic protein structures or be key components of peptide-drug conjugates. Here, we describe the development of novel synthetic strategies for two distinctive types of peptide macrobicycles. A multicomponent macrocyclo-dimerization approach is introduced for the production of interconnected β-turns, allowing two macrocyclic rings to be formed and dimerized in one pot. Also, an on-resin double stapling strategy is described for the assembly of lactam-bridged macrobicycles with stable tertiary folds.

Poly(ADP-ribosyl)ation (PARylation) is a reversible post-translational protein modification that has profound regulatory functions in metabolism, development and immunity, and is conserved throughout the eukaryotic lineage. Contrary to metazoa, many components and mechanistic details of PARylation have remained unidentified in plants. Here we present the transcriptional co-regulator RADICAL-INDUCED CELL DEATH1 (RCD1) as a plant PAR-reader. RCD1 is a multidomain protein with intrinsically disordered regions (IDRs) separating its domains. We have reported earlier that RCD1 regulates plant development and stress-tolerance by interacting with numerous transcription factors (TFs) through its C-terminal RST domain. This study suggests that the N-terminal WWE and PARP-like domains, as well as the connecting IDR play an important regulatory role for RCD1 function. We show that RCD1 binds PAR in vitro via its WWE domain and that PAR-binding determines RCD1 localization to nuclear bodies (NBs) in vivo. Additionally, we found that RCD1 function and stability is controlled by Photoregulatory Protein Kinases (PPKs). PPKs localize with RCD1 in NBs and phosphorylate RCD1 at multiple sites affecting its stability. This work proposes a mechanism for negative transcriptional regulation in plants, in which RCD1 localizes to NBs, binds TFs with its RST domain and is degraded after phosphorylation by PPKs.

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