Streamlining Protein Fractional Synthesis Rates Using SP3 Beads and Stable Isotope Mass Spectrometry: A Case Study on the Plant Ribosome.

Ribosomes are an archetypal ribonucleoprotein assembly. Due to ribosomal evolution and function, r-proteins share specific physicochemical similarities, making the riboproteome particularly suited for tailored proteome profiling methods. Moreover, the structural proteome of ribonucleoprotein assemblies reflects context-dependent functional features. Thus, characterizing the state of riboproteomes provides insights to uncover the context-dependent functionality of r-protein rearrangements, as they relate to what has been termed the ribosomal code, a concept that parallels that of the histone code, in which chromatin rearrangements influence gene expression. Compared to high-resolution ribosomal structures, omics methods lag when it comes to offering customized solutions to close the knowledge gap between structure and function that currently exists in riboproteomes. Purifying the riboproteome and subsequent shot-gun proteomics typically involves protein denaturation and digestion with proteases. The results are relative abundances of r-proteins at the ribosome population level. We have previously shown that, to gain insight into the stoichiometry of individual proteins, it is necessary to measure by proteomics bound r-proteins and normalize their intensities by the sum of r-protein abundances per ribosomal complex, i.e., 40S or 60S subunits. These calculations ensure that individual r-protein stoichiometries represent the fraction of each family/paralog relative to the complex, effectively revealing which r-proteins become substoichiometric in specific physiological scenarios. Here, we present an optimized method to profile the riboproteome of any organism as well as the synthesis rates of r-proteins determined by stable isotope-assisted mass spectrometry. Our method purifies the r-proteins in a reversibly denatured state, which offers the possibility for combined top-down and bottom-up proteomics. Our method offers a milder native denaturation of the r-proteome via a chaotropic GuHCl solution as compared with previous studies that use irreversible denaturation under highly acidic conditions to dissociate rRNA and r-proteins. As such, our method is better suited to conserve post-translational modifications (PTMs). Subsequently, our method carefully considers the amino acid composition of r-proteins to select an appropriate protease for digestion. We avoid non-specific protease cleavage by increasing the pH of our standardized r-proteome dilutions that enter the digestion pipeline and by using a digestion buffer that ensures an optimal pH for a reliable protease digestion process. Finally, we provide the R package ProtSynthesis to study the fractional synthesis rates of r-proteins. The package uses physiological parameters as input to determine peptide or protein fractional synthesis rates. Once the physiological parameters are measured, our equations allow a fair comparison between treatments that alter the biological equilibrium state of the system under study. Our equations correct peptide enrichment using enrichments in soluble amino acids, growth rates, and total protein accumulation. As a means of validation, our pipeline fails to find "false" enrichments in non-labeled samples while also filtering out proteins with multiple unique peptides that have different enrichment values, which are rare in our datasets. These two aspects reflect the accuracy of our tool. Our method offers the possibility of elucidating individual r-protein family/paralog abundances, PTM status, fractional synthesis rates, and dynamic assembly into ribosomal complexes if top-down and bottom-up proteomic approaches are used concomitantly, taking one step further into mapping the native and dynamic status of the r-proteome onto high-resolution ribosome structures. In addition, our method can be used to study the proteomes of all macromolecular assemblies that can be purified, although purification is the limiting step, and the efficacy and accuracy of the proteases may be limited depending on the digestion requirements. Key features • Efficient purification of the ribosomal proteome: streamlined procedure for the specific purification of the ribosomal proteome or complex Ome. • Accurate calculation of fractional synthesis rates: robust method for calculating fractional protein synthesis rates in macromolecular complexes under different physiological steady states. • Holistic ribosome methodology focused on plants: comprehensive approach that provides insights into the ribosomes and translational control of plants, demonstrated using cold acclimation [1]. • Tailored strategies for stable isotope labeling in plants: methodology focusing on materials and labeling considerations specific to free and proteinogenic amino acid analysis [2].

Comparative genomics points to tandem duplications of SAD gene clusters as drivers of increased α-linolenic (ω-3) content in S. hispanica seeds.

Salvia hispanica L. (chia) is a source of abundant ω-3 polyunsaturated fatty acids (ω-3-PUFAs) that are highly beneficial to human health. The genomic basis for this accrued ω-3-PUFA content in this emerging crop was investigated through the assembly and comparative analysis of a chromosome-level reference genome for S. hispanica. The highly contiguous 321.5-Mbp genome assembly covering all six chromosomes enabled the identification of 32,922 protein-coding genes. Two whole-genome duplications (WGD) events were identified in the S. hispanica lineage. However, these WGD events could not be linked to the high α-linolenic acid (ALA, ω-3) accumulation in S. hispanica seeds based on phylogenomics. Instead, our analysis supports the hypothesis that evolutionary expansion through tandem duplications of specific lipid gene families, particularly the stearoyl-acyl carrier protein desaturase (ShSAD) gene family, is the main driver of the abundance of ω-3-PUFAs in S. hispanica seeds. The insights gained from the genomic analysis of S. hispanica will help establish a molecular breeding target that can be leveraged through genome editing techniques to increase ω-3 content in oil crops.

Discovery of novel neutral glycosphingolipids in cereal crops: rapid profiling using reversed-phased HPLC-ESI-QqTOF with parallel reaction monitoring.

This study explores the sphingolipid class of oligohexosylceramides (OHCs), a rarely studied group, in barley (Hordeum vulgare L.) through a new lipidomics approach. Profiling identified 45 OHCs in barley (Hordeum vulgare L.), elucidating their fatty acid (FA), long-chain base (LCB) and sugar residue compositions; and was accomplished by monophasic extraction followed by reverse-phased high performance liquid chromatography electrospray ionisation quadrupole-time-of-flight tandem mass spectrometry (HPLC-ESI-QqTOF-MS/MS) employing parallel reaction monitoring (PRM). Results revealed unknown ceramide species and highlighted distinctive FA and LCB compositions when compared to other sphingolipid classes. Structurally, the OHCs featured predominantly trihydroxy LCBs associated with hydroxylated FAs and oligohexosyl residues consisting of two-five glucose units in a linear 1 → 4 linkage. A survey found OHCs in tissues of major cereal crops while noting their absence in conventional dicot model plants. This study found salinity stress had only minor effects on the OHC profile in barley roots, leaving questions about their precise functions in plant biology unanswered.

Leaf metabolic traits reveal hidden dimensions of plant form and function.

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.

Lipid remodeling of contrasting maize (Zea mays L.) hybrids under repeated drought.

The role of recovery after drought has been proposed to play a more prominent role during the whole drought-adaption process than previously thought. Two maize hybrids with comparable growth but contrasting physiological responses were investigated using physiological, metabolic, and lipidomic tools to understand the plants' strategies of lipid remodeling in response to repeated drought stimuli. Profound differences in adaptation between hybrids were discovered during the recovery phase, which likely gave rise to different degrees of lipid adaptability to the subsequent drought event. These differences in adaptability are visible in galactolipid metabolism and fatty acid saturation patterns during recovery and may lead to a membrane dysregulation in the sensitive maize hybrid. Moreover, the more drought-tolerant hybrid displays more changes of metabolite and lipid abundance with a higher number of differences within individual lipids, despite a lower physiological response, while the responses in the sensitive hybrid are higher in magnitude but lower in significance on the level of individual lipids and metabolites. This study suggests that lipid remodeling during recovery plays a key role in the drought response of plants.

Molecular Mechanisms of Pseudomonas-Assisted Plant Nitrogen Uptake: Opportunities for Modern Agriculture.

Pseudomonas spp. make up 1.6% of the bacteria in the soil and are found throughout the world. More than 140 species of this genus have been identified, some beneficial to the plant. Several species in the family Pseudomonadaceae, including Azotobacter vinelandii AvOP, Pseudomonas stutzeri A1501, Pseudomonas stutzeri DSM4166, Pseudomonas szotifigens 6HT33bT, and Pseudomonas sp. strain K1 can fix nitrogen from the air. The genes required for these reactions are organized in a nitrogen fixation island, obtained via horizontal gene transfer from Klebsiella pneumoniae, Pseudomonas stutzeri, and Azotobacter vinelandii. Today, this island is conserved in Pseudomonas spp. from different geographical locations, which, in turn, have evolved to deal with different geo-climatic conditions. Here, we summarize the molecular mechanisms behind Pseudomonas-driven plant growth promotion, with particular focus on improving plant performance at limiting nitrogen (N) and improving plant N content. We describe Pseudomonas-plant interaction strategies in the soil, noting that the mechanisms of denitrification, ammonification, and secondary metabolite signaling are only marginally explored. Plant growth promotion is dependent on the abiotic conditions and differs at sufficient and deficient N. The molecular controls behind different plant responses are not fully elucidated. We suggest that superposition of transcriptome, proteome, and metabolome data and their integration with plant phenotype development through time will help fill these gaps. The aim of this review is to summarize the knowledge behind Pseudomonas-driven nitrogen fixation and to point to possible agricultural solutions. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

Plant Growth Promotion and Heat Stress Amelioration in Arabidopsis Inoculated with Paraburkholderia phytofirmans PsJN Rhizobacteria Quantified with the GrowScreen-Agar II Phenotyping Platform.

High temperatures inhibit plant growth. A proposed strategy for improving plant productivity under elevated temperatures is the use of plant growth-promoting rhizobacteria (PGPR). While the effects of PGPR on plant shoots have been extensively explored, roots-particularly their spatial and temporal dynamics-have been hard to study, due to their below-ground nature. Here, we characterized the time- and tissue-specific morphological changes in bacterized plants using a novel non-invasive high-resolution plant phenotyping and imaging platform-GrowScreen-Agar II. The platform uses custom-made agar plates, which allow air exchange to occur with the agar medium and enable the shoot to grow outside the compartment. The platform provides light protection to the roots, the exposure of it to the shoots, and the non-invasive phenotyping of both organs. Arabidopsis thaliana, co-cultivated with Paraburkholderia phytofirmans PsJN at elevated and ambient temperatures, showed increased lengths, growth rates, and numbers of roots. However, the magnitude and direction of the growth promotion varied depending on root type, timing, and temperature. The root length and distribution per depth and according to time was also influenced by bacterization and the temperature. The shoot biomass increased at the later stages under ambient temperature in the bacterized plants. The study offers insights into the timing of the tissue-specific, PsJN-induced morphological changes and should facilitate future molecular and biochemical studies on plant-microbe-environment interactions.

Measures of insulin sensitivity, leptin, and adiponectin concentrations in cats in diabetic remission compared to healthy control cats.

Objectives: Firstly, to compare differences in insulin, adiponectin, leptin, and measures of insulin sensitivity between diabetic cats in remission and healthy control cats, and determine whether these are predictors of diabetic relapse. Secondly, to determine if these hormones are associated with serum metabolites known to differ between groups. Thirdly, if any of the hormonal or identified metabolites are associated with measures of insulin sensitivity. Animals: Twenty cats in diabetic remission for a median of 101 days, and 21 healthy matched control cats. Methods: A casual blood glucose measured on admission to the clinic. Following a 24 h fast, a fasted blood glucose was measured, and blood sample taken for hormone (i.e., insulin, leptin, and adiponectin) and untargeted metabolomic (GC-MS and LC-MS) analysis. A simplified IVGGT (1 g glucose/kg) was performed 3 h later. Cats were monitored for diabetes relapse for at least 9 months (270 days). Results: Cats in diabetic remission had significantly higher serum glucose and insulin concentrations, and decreased insulin sensitivity as indicated by an increase in HOMA and decrease in QUICKI and Bennett indices. Leptin was significantly increased, but there was no difference in adiponectin (or body condition score). Several significant correlations were found between insulin sensitivity indices, leptin, and serum metabolites identified as significantly different between remission and control cats. No metabolites were significantly correlated with adiponectin. No predictors of relapse were identified in this study. Conclusion and clinical importance: Insulin resistance, an underlying factor in diabetic cats, persists in diabetic remission. Cats in remission should be managed to avoid further exacerbating insulin resistance.

Biochemical Changes in Two Barley Genotypes Inoculated With a Beneficial Fungus Trichoderma harzianum Rifai T-22 Grown in Saline Soil.

One of the most important environmental factors impacting crop plant productivity is soil salinity. Fungal endophytes have been characterised as biocontrol agents that help in plant productivity and induce resistance responses to several abiotic stresses, including salinity. In the salt-tolerant cereal crop barley (Hordeum vulgare L.), there is limited information about the metabolites and lipids that change in response to inoculation with fungal endophytes in saline conditions. In this study, gas chromatography coupled to mass spectrometry (GC-MS) and LC-electrospray ionisation (ESI)-quadrupole-quadrupole time of flight (QqTOF)-MS were used to determine the metabolite and lipid changes in two fungal inoculated barley genotypes with differing tolerance levels to saline conditions. The more salt-tolerant cultivar was Vlamingh and less salt tolerant was Gairdner. Trichoderma harzianum strain T-22 was used to treat these plants grown in soil under control and saline (200 mM NaCl) conditions. For both genotypes, fungus-colonised plants exposed to NaCl had greater root and shoot biomass, and better chlorophyll content than non-colonised plants, with colonised-Vlamingh performing better than uninoculated control plants. The metabolome dataset using GC-MS consisted of a total of 93 metabolites of which 74 were identified in roots of both barley genotypes as organic acids, sugars, sugar acids, sugar alcohols, amino acids, amines, and a small number of fatty acids. LC-QqTOF-MS analysis resulted in the detection of 186 lipid molecular species, classified into three major lipid classes-glycerophospholipids, glycerolipids, and sphingolipids, from roots of both genotypes. In Cultivar Vlamingh both metabolites and lipids increased with fungus and salt treatment while in Gairdner they decreased. The results from this study suggest that the metabolic pathways by which the fungus imparts salt tolerance is different for the different genotypes.

Germline mutations in mitochondrial complex I reveal genetic and targetable vulnerability in IDH1-mutant acute myeloid leukaemia.

The interaction of germline variation and somatic cancer driver mutations is under-investigated. Here we describe the genomic mitochondrial landscape in adult acute myeloid leukaemia (AML) and show that rare variants affecting the nuclear- and mitochondrially-encoded complex I genes show near-mutual exclusivity with somatic driver mutations affecting isocitrate dehydrogenase 1 (IDH1), but not IDH2 suggesting a unique epistatic relationship. Whereas AML cells with rare complex I variants or mutations in IDH1 or IDH2 all display attenuated mitochondrial respiration, heightened sensitivity to complex I inhibitors including the clinical-grade inhibitor, IACS-010759, is observed only for IDH1-mutant AML. Furthermore, IDH1 mutant blasts that are resistant to the IDH1-mutant inhibitor, ivosidenib, retain sensitivity to complex I inhibition. We propose that the IDH1 mutation limits the flexibility for citrate utilization in the presence of impaired complex I activity to a degree that is not apparent in IDH2 mutant cells, exposing a mutation-specific metabolic vulnerability. This reduced metabolic plasticity explains the epistatic relationship between the germline complex I variants and oncogenic IDH1 mutation underscoring the utility of genomic data in revealing metabolic vulnerabilities with implications for therapy.

The Effect of Cold Stress on the Root-Specific Lipidome of Two Wheat Varieties with Contrasting Cold Tolerance.

Complex glycerolipidome analysis of wheat upon low temperature stress has been reported for above-ground tissues only. There are no reports on the effects of cold stress on the root lipidome nor on tissue-specific responses of cold stress wheat roots. This study aims to investigate the changes of lipid profiles in the different developmental zones of the seedling roots of two wheat varieties with contrasting cold tolerance exposed to chilling and freezing temperatures. We analyzed 273 lipid species derived from 21 lipid classes using a targeted profiling approach based on MS/MS data acquired from schedule parallel reaction monitoring assays. For both the tolerant Young and sensitive Wyalkatchem species, cold stress increased the phosphatidylcholine and phosphatidylethanolamine compositions, but decreased the monohexosyl ceramide compositions in the root zones. We show that the difference between the two varieties with contrasting cold tolerance could be attributed to the change in the individual lipid species, rather than the fluctuation of the whole lipid classes. The outcomes gained from this study may advance our understanding of the mechanisms of wheat adaptation to cold and contribute to wheat breeding for the improvement of cold-tolerance.

Salt stress alters membrane lipid content and lipid biosynthesis pathways in the plasma membrane and tonoplast.

Plant cell membranes are the sites of sensing and initiation of rapid responses to changing environmental factors including salinity stress. Understanding the mechanisms involved in membrane remodeling is important for studying salt tolerance in plants. This task remains challenging in complex tissue due to suboptimal subcellular membrane isolation techniques. Here, we capitalized on the use of a surface charge-based separation method, free flow electrophoresis, to isolate the tonoplast (TP) and plasma membrane (PM) from leaf tissue of the halophyte ice plant (Mesembryanthemum crystallinum L.). Results demonstrated a membrane-specific lipidomic remodeling in this plant under salt conditions, including an increased proportion of bilayer forming lipid phosphatidylcholine in the TP and an increase in nonbilayer forming and negatively charged lipids (phosphatidylethanolamine and phosphatidylserine) in the PM. Quantitative proteomics showed salt-induced changes in proteins involved in fatty acid synthesis and desaturation, glycerolipid, and sterol synthesis, as well as proteins involved in lipid signaling, binding, and trafficking. These results reveal an essential plant mechanism for membrane homeostasis wherein lipidome remodeling in response to salt stress contributes to maintaining the physiological function of individual subcellular compartments.

Low doses of the organic insecticide spinosad trigger lysosomal defects, elevated ROS, lipid dysregulation, and neurodegeneration in flies.

Large-scale insecticide application is a primary weapon in the control of insect pests in agriculture. However, a growing body of evidence indicates that it is contributing to the global decline in population sizes of many beneficial insect species. Spinosad emerged as an organic alternative to synthetic insecticides and is considered less harmful to beneficial insects, yet its mode of action remains unclear. Using Drosophila, we show that low doses of spinosad antagonize its neuronal target, the nicotinic acetylcholine receptor subunit alpha 6 (nAChRα6), reducing the cholinergic response. We show that the nAChRα6 receptors are transported to lysosomes that become enlarged and increase in number upon low doses of spinosad treatment. Lysosomal dysfunction is associated with mitochondrial stress and elevated levels of reactive oxygen species (ROS) in the central nervous system where nAChRα6 is broadly expressed. ROS disturb lipid storage in metabolic tissues in an nAChRα6-dependent manner. Spinosad toxicity is ameliorated with the antioxidant N-acetylcysteine amide. Chronic exposure of adult virgin females to low doses of spinosad leads to mitochondrial defects, severe neurodegeneration, and blindness. These deleterious effects of low-dose exposures warrant rigorous investigation of its impacts on beneficial insects.

Metabolomics as an emerging tool to study plant-microbe interactions.

In natural environments, interaction between plant roots and microorganisms are common. These interactions between microbial species and plants inhabited by them are being studied using various techniques. Metabolomics research based on mass spectrometric techniques is one of the crucial approaches that underpins system biology and relies on precision instrument analysis. In the last decade, this emerging field has received extensive attention. It provides a qualitative and quantitative approach for determining the mechanisms of symbiosis of bacteria and fungi with plants and also helps to elucidate the tolerance mechanisms of host plants against various abiotic stresses. However, this -omics application and its tools in plant-microbe interaction studies is still underutilized compared with genomic and transcriptomic methods. Therefore, it is crucial to bring this field forward to bear on the study of plant resistance and susceptibility. This review describes the current status of methods and progress in metabolomics applications for plant-microbe interaction studies discussing current challenges and future prospects.

Modulators or facilitators? Roles of lipids in plant root-microbe interactions.

Lipids have diverse functions in regulating the plasma membrane's cellular processes and signaling mediation. Plasma membrane lipids are also involved in the plant's complex interactions with the surrounding microorganisms, with which plants are in various forms of symbiosis. The roles of lipids influence the whole microbial colonization process, thus shaping the rhizomicrobiome. As chemical signals, lipids facilitate the stages of rhizospheric interactions - from plant root to microbe, microbe to microbe, and microbe to plant root - and modulate the plant's defense responses upon perception or contact with either beneficial or phytopathogenic microorganisms. Although studies have come a long way, further investigation is needed to discover more lipid species and elucidate novel lipid functions and profiles under various stages of plant root-microbe interactions.

Next-generation enhanced-efficiency fertilizers for sustained food security.

Nitrogen losses in agricultural systems can be reduced through enhanced-efficiency fertilizers (EEFs), which control the physicochemical release from fertilizers and biological nitrogen transformations in soils. The adoption of EEFs by farmers requires evidence of consistent performance across soils, crops and climates, paired with information on the economic advantages. Here we show that the benefits of EEFs due to avoided social costs of nitrogen pollution considerably outweigh their costs-and must be incorporated in fertilizer policies. We outline new approaches to the design of EEFs using enzyme inhibitors with modifiable chemical structures and engineered, biodegradable coatings that respond to plant rhizosphere signalling molecules.