High-throughput Comparison, Functional Annotation, and Metabolic Modeling of Plant Genomes using the PlantSEED Resource

There is a growing demand for genome-scale metabolic reconstructions for plants, fueled by the need to understand the metabolic basis of crop yield and by progress in genome and transcriptome sequencing. Methods are also required to enable the interpretation of plant transcriptome data to study how cellular metabolic activity varies under different growth conditions or even within different organs, tissues, and developmental stages. Such methods depend extensively on the accuracy with which genes have been mapped to the biochemical reactions in the plant metabolic pathways. Errors in these mappings lead to metabolic reconstructions with an inflated number of reactions and possible generation of unreliable metabolic phenotype predictions. Here we introduce a new evidence-based genome-scale metabolic reconstruction of maize, with significant improvements in the quality of the gene-reaction associations included within our model. We also present a new approach for applying our model to predict active metabolic genes based on transcriptome data. This method includes a minimal set of reactions associated with low expression genes to enable activity of a maximum number of reactions associated with high expression genes. We apply this method to construct an organ-specific model for the maize leaf, and tissue specific models for maize embryo and endosperm cells. We validate our models using fluxomics data for the endosperm and embryo, demonstrating an improved capacity of our models to fit the available fluxomics data. All models are publicly available via the DOE Systems Biology Knowledgebase and PlantSEED, and our new method is generally applicable for analysis transcript profiles from any plant, paving the way for further in silico studies with a wide variety of plant genomes

Identification and characterization of a lipase expressed during Arabidopsis thaliana reserves hydrolysis

Les réserves d’huile de la graine d’Arabidopsis thaliana sont hydrolysées par des lipases au cours de la croissance post-germinative de la plantule. Une protéine capable de fixer un inhibiteur de lipase a été identifiée à partir d’un extrait de plantules de colza. La séquence de cette protéine ressemble à celles de lipases connues. L’expression transitoire du gène orthologue d’Arabidopsis chez Nicotiana benthamiana induit l’apparition d’une activité lipase. Ces données suggèrent que cette protéine est une lipase. Une étude de la localisation in vivo de cette enzyme chez Nicotiana benthamiana indique qu’elle est localisée au niveau des peroxysomes. Chez Arabidopsis, le gène codant pour cette lipase est exprimé essentiellement lors de la croissance des plantules, quand l’hydrolyse de l’huile est maximale. L’analyse d’un mutant montre que ce gène est responsable de l’essentiel de l’activité lipase mesurée pendant la mobilisation de l’huile de réserve. Ces données suggèrent que cette lipase pourrait être impliquée dans la mobilisation des réserves lipidiques pendant la croissance post-germinative. Néanmoins, l’hydrolyse des réserves n’est pas diminuée chez le mutant. Cela pourrait être lié à une compensation par d’autres lipases.In germinating seedlings of Arabidopsis thaliana, fat storage breakdown is initiated by lipases. A protein capable to bind to a lipase inhibitor was identified from an extract of rape seedlings and its amino acid sequence found to resemble that of known lipases. Transient expression of the Arabidopsis orthologous gene led to a 100-fold increase in lipase activity in Nicotiana bethamiana leaves. Taken together, these data strongly suggest that this protein is indeed a lipase. In vivo localization studies using a GFP fusion protein in Nicotiana benthamiana as a transcient expression host showed a peroxisomal localization. In Arabidopsis, the gene coding for this lipase was found to be mainly expressed in seedlings during fat storage breakdown. Most lipase activity was abolished in germinating seedlings of an Arabidopsis mutant for this gene. These data suggest that this lipase is likely involved in the breakdown of fat storage in germinating seedlings of Arabidopsis. However, oil mobilization was not affected in Arabidopsis mutant plants. This might suggest that the effect of the mutation could be compensated for by other lipases

Functional Annotations of Paralogs: A Blessing and a Curse

Gene duplication followed by mutation is a classic mechanism of neofunctionalization, producing gene families with functional diversity. In some cases, a single point mutation is sufficient to change the substrate specificity and/or the chemistry performed by an enzyme, making it difficult to accurately separate enzymes with identical functions from homologs with different functions. Because sequence similarity is often used as a basis for assigning functional annotations to genes, non-isofunctional gene families pose a great challenge for genome annotation pipelines. Here we describe how integrating evolutionary and functional information such as genome context, phylogeny, metabolic reconstruction and signature motifs may be required to correctly annotate multifunctional families. These integrative analyses can also lead to the discovery of novel gene functions, as hints from specific subgroups can guide the functional characterization of other members of the family. We demonstrate how careful manual curation processes using comparative genomics can disambiguate subgroups within large multifunctional families and discover their functions. We present the COG0720 protein family as a case study. We also discuss strategies to automate this process to improve the accuracy of genome functional annotation pipelines

Identification and characterization of a lipase expressed during Arabidopsis thaliana reserves hydrolysis

Les réserves d’huile de la graine d’Arabidopsis thaliana sont hydrolysées par des lipases au cours de la croissance post-germinative de la plantule. Une protéine capable de fixer un inhibiteur de lipase a été identifiée à partir d’un extrait de plantules de colza. La séquence de cette protéine ressemble à celles de lipases connues. L’expression transitoire du gène orthologue d’Arabidopsis chez Nicotiana benthamiana induit l’apparition d’une activité lipase. Ces données suggèrent que cette protéine est une lipase. Une étude de la localisation in vivo de cette enzyme chez Nicotiana benthamiana indique qu’elle est localisée au niveau des peroxysomes. Chez Arabidopsis, le gène codant pour cette lipase est exprimé essentiellement lors de la croissance des plantules, quand l’hydrolyse de l’huile est maximale. L’analyse d’un mutant montre que ce gène est responsable de l’essentiel de l’activité lipase mesurée pendant la mobilisation de l’huile de réserve. Ces données suggèrent que cette lipase pourrait être impliquée dans la mobilisation des réserves lipidiques pendant la croissance post-germinative. Néanmoins, l’hydrolyse des réserves n’est pas diminuée chez le mutant. Cela pourrait être lié à une compensation par d’autres lipases.In germinating seedlings of Arabidopsis thaliana, fat storage breakdown is initiated by lipases. A protein capable to bind to a lipase inhibitor was identified from an extract of rape seedlings and its amino acid sequence found to resemble that of known lipases. Transient expression of the Arabidopsis orthologous gene led to a 100-fold increase in lipase activity in Nicotiana bethamiana leaves. Taken together, these data strongly suggest that this protein is indeed a lipase. In vivo localization studies using a GFP fusion protein in Nicotiana benthamiana as a transcient expression host showed a peroxisomal localization. In Arabidopsis, the gene coding for this lipase was found to be mainly expressed in seedlings during fat storage breakdown. Most lipase activity was abolished in germinating seedlings of an Arabidopsis mutant for this gene. These data suggest that this lipase is likely involved in the breakdown of fat storage in germinating seedlings of Arabidopsis. However, oil mobilization was not affected in Arabidopsis mutant plants. This might suggest that the effect of the mutation could be compensated for by other lipases

EFI-EST, EFI-GNT, and EFI-CGFP: Enzyme Function Initiative (EFI) Web Resource for Genomic Enzymology Tools

The Enzyme Function Initiative (EFI) provides a web resource with "genomic enzymology" web tools to leverage the protein (UniProt) and genome (European Nucleotide Archive; ENA; https://www.ebi.ac.uk/ena/) databases to assist the assignment of in vitro enzymatic activities and in vivo metabolic functions to uncharacterized enzymes (https://efi.igb.illinois.edu/). The tools enable (1) exploration of sequence-function space in enzyme families using sequence similarity networks (SSNs; EFI-EST), (2) easy access to genome context for bacterial, archaeal, and fungal proteins in the SSN clusters so that isofunctional families can be identified and their functions inferred from genome context (EFI-GNT); and (3) determination of the abundance of SSN clusters in NIH Human Metagenome Project metagenomes using chemically guided functional profiling (EFI-CGFP). We describe enhancements that enable SSNs to be generated from taxonomy categories, allowing higher resolution analyses of sequence-function space; we provide examples of the generation of taxonomy category-specific SSNs

Polyphosphoinositides Are Enriched in Plant Membrane Rafts and Form Microdomains in the Plasma Membrane1[W]

In this article, we analyzed the lipid composition of detergent-insoluble membranes (DIMs) purified from tobacco (Nicotiana tabacum) plasma membrane (PM), focusing on polyphosphoinositides, lipids known to be involved in various signal transduction events. Polyphosphoinositides were enriched in DIMs compared with whole PM, whereas all structural phospholipids were largely depleted from this fraction. Fatty acid composition analyses suggest that enrichment of polyphosphoinositides in DIMs is accompanied by their association with more saturated fatty acids. Using an immunogold-electron microscopy strategy, we were able to visualize domains of phosphatidylinositol 4,5-bisphosphate in the plane of the PM, with 60% of the epitope found in clusters of approximately 25 nm in diameter and 40% randomly distributed at the surface of the PM. Interestingly, the phosphatidylinositol 4,5-bisphosphate cluster formation was not significantly sensitive to sterol depletion induced by methyl-β-cyclodextrin. Finally, we measured the activities of various enzymes of polyphosphoinositide metabolism in DIMs and PM and showed that these activities are present in the DIM fraction but not enriched. The putative role of plant membrane rafts as signaling membrane domains or membrane-docking platforms is discussed

Biosynthesis and function of 7-deazaguanine derivatives in bacteria and phages

Deazaguanine modifications play multifaceted roles in the molecular biology of DNA and tRNA, shaping diverse yet essential biological processes, including the nuanced fine-tuning of translation efficiency and the intricate modulation of codon-anticodon interactions. Beyond their roles in translation, deazaguanine modifications contribute to cellular stress resistance, self-nonself discrimination mechanisms, and host evasion defenses, directly modulating the adaptability of living organisms. Deazaguanine moieties extend beyond nucleic acid modifications, manifesting in the structural diversity of biologically active natural products. Their roles in fundamental cellular processes and their presence in biologically active natural products underscore their versatility and pivotal contributions to the intricate web of molecular interactions within living organisms. Here, we discuss the current understanding of the biosynthesis and multifaceted functions of deazaguanines, shedding light on their diverse and dynamic roles in the molecular landscape of life

Cryptic enzymatic assembly of peptides armed with β-lactone warheads

Nature has evolved biosynthetic pathways to molecules possessing reactive warheads that inspired the development of many therapeutic agents, including penicillin antibiotics. Peptides armed with electrophilic warheads have proven to be particularly effective covalent inhibitors, providing essential antimicrobial, antiviral and anticancer agents. Here we provide a full characterization of the pathways that nature deploys to assemble peptides with β-lactone warheads, which are potent proteasome inhibitors with promising anticancer activity. Warhead assembly involves a three-step cryptic methylation sequence, which is likely required to reduce unfavorable electrostatic interactions during the sterically demanding β-lactonization. Amide-bond synthetase and adenosine triphosphate (ATP)-grasp enzymes couple amino acids to the β-lactone warhead, generating the bioactive peptide products. After reconstituting the entire pathway to β-lactone peptides in vitro, we go on to deliver a diverse range of analogs through enzymatic cascade reactions. Our approach is more efficient and cleaner than the synthetic methods currently used to produce clinically important warhead-containing peptides.</p