Highly efficient phosphorus (P)-use strategies have evolved in plants, allowing them to thrive in severely P-impoverished environments. However, it remains unclear how allocating leaf P to biochemical fractions, including specific P-containing metabolites, contributes to instantaneous photosynthetic P-use efficiency (iPPUE) and the position of species along the leaf economics spectrum (LES). We measured LES-associated traits (leaf mass per area, light-saturated photosynthetic rate, and P and nitrogen concentrations) and iPPUE in 12 coexisting Proteaceae, Fabaceae, Myrtaceae and Ericaceae from two severely P-impoverished soils. We characterised patterns of P allocation into lipids, nucleic acids, metabolites, a residual fraction and inorganic P, and identified P-containing metabolites within the metabolite-P fraction. We identified multiple P-utilisation strategies that were both species-dependent and soil context-dependent. The iPPUE and LES scores, representing leaf-level resource-use strategies, were positively associated with P concentrations in nucleic acids and the residual P fraction. Concentrations of key P-containing metabolites of the Calvin-Benson cycle, ribulose-1,5-bisphosphate and 3-phosphoglycerate, were positively correlated with and served as strong predictors of iPPUE and resource-use strategies within the LES. Species in severely P-impoverished habitats exhibited species-dependent and soil context-dependent P-allocation patterns and maintained specific P-containing metabolite concentrations. These trait combinations underpin high iPPUE and enable multiple resource-use strategies.

Oat (Avena sativa L.) is an important cereal grain with a unique nutritional profile including a high proportion of lipids. Understanding lipid composition and distribution in oats is valuable for plant, food and nutritional research, and can be achieved using MALDI mass spectrometry imaging (MALDI-MSI). However, this approach presents several challenges for sample preparation (hardness of grains) and analysis (isobaric and isomeric properties of lipids). Here, oat sections were successfully mounted onto gelatin-coated indium tin oxide slides with minimal tearing. Poor detection of triacylglycerols was resolved by applying sodium chloride during mounting, increasing signal intensity. In combination with trapped ion mobility spectrometry (TIMS), lipid identification significantly improved, and we report the separation of several isobaric and isomeric lipids with visualisation of their "true" spatial distributions. This study describes a novel MALDI-TIMS-MSI analytical technique for oat lipids, which may be used to improve the discovery of biomarkers for grain quality.

Understanding the systemic biochemistry of early pregnancy in the mare is essential for developing new diagnostics and identifying causes for pregnancy loss. This study aimed to elucidate the dynamic lipidomic changes occurring during the initial stages of equine pregnancy, with a specific focus on days 7 and 14 post-ovulation. By analysing and comparing the plasma lipid profiles of pregnant and non-pregnant mares, the objective of this study was to identify potential biomarkers for pregnancy and gain insights into the biochemical adaptations essential for supporting maternal recognition of pregnancy and early embryonic development. Employing discovery lipidomics, we analysed plasma samples from pregnant and non-pregnant mares on days 7 and 14 post-conception using the SCIEX ZenoTOF 7600 system. This high-resolution mass spectrometry approach enabled us to comprehensively profile and compare the lipidomes across these critical early gestational timepoints. Our analysis revealed significant lipidomic alterations between pregnant and non-pregnant mares and between days 7 and 14 of pregnancy. Key findings include the upregulation of bile acids, sphingomyelins, phosphatidylinositols, and triglycerides in pregnant mares. These changes suggest enhanced lipid synthesis and mobilization, likely associated with the embryo's nutritional requirements and the establishment of embryo-maternal interactions. There were significant differences in lipid metabolism between pregnant and non-pregnant mares, with a notable increase in the sterol lipid BA 24:1;O5 in pregnant mares as early as day 7 of gestation, suggesting it as a sensitive biomarker for early pregnancy detection. Notably, the transition from day 7 to day 14 in pregnant mares is characterized by a shift towards lipids indicative of membrane biosynthesis, signalling activity, and preparation for implantation. The study demonstrates the profound lipidomic shifts that occur in early equine pregnancy, highlighting the critical role of lipid metabolism in supporting embryonic development. These findings provide valuable insights into the metabolic adaptations during these period and potential biomarkers for early pregnancy detection in mares.

Analysis of salinity tolerance processes in wheat has focused on salt exclusion from shoots while root phenotypes have received limited attention. Here, we consider the varying phenotypic response of four bread wheat varieties that differ in their type and degree of salt tolerance and assess their molecular responses to salinity and changes in root cell wall lignification. These varieties were Westonia introgressed with Nax1 and Nax2 root sodium transporters (HKT1;4-A and HKT1;5-A) that reduce Na+ accumulation in leaves, as well as the 'tissue tolerant' Portuguese landrace Mocho de Espiga Branca that has a mutation in the homologous gene HKT1;5-D and has high Na+ concentration in leaves. These three varieties were compared with the relatively more salt-sensitive cultivar Gladius. Through the use of root histochemical analysis, ion concentrations, as well as differential proteomics and targeted metabolomics, we provide an integrated view of the wheat root response to salinity. We show different metabolic re-arrangements in energy conversion, primary metabolic machinery and phenylpropanoid pathway leading to monolignol production in a genotype and genotype by treatment-dependent manner that alters the extent and localisation of root lignification which correlated with an improved capacity of wheat roots to cope better under salinity stress.

Increases in soil salinity impact growth and agricultural yield by inhibiting plant functions. Interactions of fungal endophytes with crop plants can improve tolerance and is a less expensive approach. Here, the role of Trichoderma harzianum T-22 in alleviating NaCl-induced stress in two barley genotypes (cv. Vlamingh and cv. Gairdner) has been investigated. Metabolomics using GC-MS for polar metabolites and LC-MS for lipids was employed to provide insights into the biochemical changes in inoculated roots during the early stages of interaction. T. harzianum improved root growth of both genotypes in saline conditions whereas uninoculated roots were significantly shorter after salt treatment. The fungus reduced relative concentration of sugars in both genotypes under saline conditions but there was no change in organic acids. Amino acids decreased only in cv. Gairdner in fungus-inoculated roots under saline conditions. Lipid analysis suggested that salt stress causes large changes in roots but that inoculation with fungus greatly reduces the extent of these changes. By studying a tolerant and a sensitive genotype and their responses to salt and inoculation we have been able to develop hypotheses that explain the tolerance of Vlamingh to salt and how fungal inoculation changes the response of Gairdner to improve its tolerance.

Identifying the molecular basis of thermotolerance in crops is becoming increasingly important with the changing climatic conditions that challenge future food security. Sustaining cellular energy production under heat stress is vital in maintaining an uninterrupted growth cycle, and thus the mitochondria is instrumental in facilitating the overall heat-tolerance of a crop plant. Using targeted mass spectrometry, the changes in abundance of the lipo-protein network in mitochondrial membranes following a short episode of extremely high temperature were analysed in two wheat cultivars of differing thermotolerance. The results indicated that membrane lipids remodel in favour of shorter fatty acyl tails, and an increase in the abundance of phosphatidylinositol, while specific to the heat-tolerant cultivar was an increase in the abundance of phosphatidylserine. The differences between the lipid profiles of the two cultivars is a likely explanation for the decrease in Complex I NADH dehydrogenase activity in the heat-sensitive cultivar. Further metabolite analysis by LC-MS revealed malate accumulation, indicating that the disruption in Complex I activity impacts the catabolism of reducing equivalents. The measured increase in the total amount of phosphatidylserine in the heat-tolerant cultivar suggests a potential role in conveying thermotolerance for this minor membrane constituent, and highlights that a focus on membrane lipid composition during thermal stress will be essential for the breeding of future heat tolerant crops.Summary ### Competing Interest StatementThe authors have declared no competing interest.

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.

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.

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.

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.

Soil salinity has a serious impact on plant growth and agricultural yield. Inoculation of crop plants with fungal endophytes is a cost-effective way to improve salt tolerance. We used metabolomics to study how Trichoderma harzianum T-22 alleviates NaCl-induced stress in two barley (Hordeum vulgare L.) cultivars, Gairdner and Vlamingh, with contrasting salinity tolerance. GC-MS was used to analyse polar metabolites and LC-MS to analyse lipids in roots during the early stages of interaction with Trichoderma. Inoculation reversed the severe effects of salt on root length in sensitive cv. Gairdner and, to a lesser extent, improved root growth in more tolerance cv. Vlamingh. Biochemical changes showed a similar pattern in inoculated roots after salt treatment. Sugars increased in both cultivars, with ribulose, ribose, and rhamnose specifically increased by inoculation. Salt stress caused large changes in lipids in roots but inoculation with fungus greatly reduced the extent of these changes. Many of the metabolic changes in inoculated cv. Gairdner after salt treatment mirror the response of uninoculated cv. Vlamingh, but there are some metabolites that changed in both cultivars only after fungal inoculation. Further study is required to determine how these metabolic changes are induced by fungal inoculation.

Metabolic profiling of Pratylenchus thornei resistant and susceptible wheat genotypes indicates that fatty acid, glycerolipid and flavonoid classes of metabolites constitutively expressed in resistant wheat roots could reduce nematode reproduction. The root-lesion nematode Pratylenchus thornei reduces wheat production in many parts of the world. In this study the metabolic profiles of two wheat genotypes ‘QT16258’ (moderately resistant) and ‘Janz’ (susceptible) were compared at 8 weeks post inoculation with or without P. thornei. We performed untargeted liquid chromatography mass spectrometry analysis (LC–MS) of the wheat root samples. A total of 11,704 MS features were identified, out of which 765 MS features were annotated using in-house chemical standards. Principal components analysis (PCA) and partial least square discriminant analysis (PLS-DA) indicated dissimilarity of the metabolome between P. thornei resistant and susceptible genotypes. Two-way analysis of variance indicated that metabolic differences were mainly constitutive rather than induced by inoculation with P. thornei. Eighty-four annotated metabolites were significantly (p ≤ 0.01) higher in relative concentration in ‘QT16258’ than ‘Janz’ and belonged to the following classes of metabolites: flavonoids, fatty acids, glycerolipids, alkaloids, tannins, nucleotides, steroid glycosides and terpenoids. Eighty-five annotated metabolites were significantly (p ≤ 0.01) higher in relative concentration in ‘Janz’ than ‘QT16258’ and belonged to the following classes of metabolites: amino acids, sugars, flavonoids and alkaloids. Several metabolites at higher concentration in ‘QT16258’, including quercetin-3,4'-O-di-beta-glucoside (flavonoid), linoleic acid (fatty acid), lysophosphatidylethanolamine (glycerolipid), hirsutine (alkaloid), 1-methylsulfinylbutenyl-isothiocyanate (glucosinolate), could potentially strengthen the root cell walls to inhibit nematode penetration and/or reduce nematode motility. Some metabolites at higher concentrations in susceptible ‘Janz’, including phenolics, coniferyl alcohol and indole acetic acid conjugates, could be nematode attractants as well as part of a hypersensitive browning reaction to nematode invasion.

Summary Mass spectrometry is the predominant analytical tool used in the field of plant lipidomics. However, there are many challenges associated with the mass spectrometric detection and identification of lipids because of the highly complex nature of plant lipids. Studies into lipid biosynthetic pathways, gene functions in lipid metabolism, lipid changes during plant growth and development, and the holistic examination of the role of plant lipids in environmental stress responses are often hindered. Here, we leveraged a robust pipeline that we previously established to extract and analyze lipid profiles of different tissues and developmental stages from the model plant Arabidopsis thaliana. We analyzed seven tissues at several different developmental stages and identified more than 200 lipids from each tissue analyzed. The data were used to create a web‐accessible in silico lipid map that has been integrated into an electronic Fluorescent Pictograph (eFP) browser. This in silico library of Arabidopsis lipids allows the visualization and exploration of the distribution and changes of lipid levels across selected developmental stages. Furthermore, it provides information on the characteristic fragments of lipids and adducts observed in the mass spectrometer and their retention times, which can be used for lipid identification. The Arabidopsis tissue lipid map can be accessed at http://bar.utoronto.ca/efp_arabidopsis_lipid/cgi‐bin/efpWeb.cgi.

Lipids are a group of compounds with diverse structures that perform several important functions in plants. To unravel and better understand their in vivo functions, plant biologists have been using various lipidomic technologies including liquid-chromatography (LC)-mass spectrometry (MS). However, there are still significant challenges in LC-MS based plant lipidomics, which need to be addressed. In this review, we provide an overview of the key developments in LC-MS based lipidomic approaches to detect and identify plant lipids with emphasis on areas that can be further improved. Given that the cellular lipidome is estimated to contain hundreds of thousands of lipids,1,2 many of the lipid structures remain to be discovered. Furthermore, the plant lipidome is considered to be significantly more complex compared to that of mammals. Recent technical developments in mass spectrometry have made the detection of novel lipids possible; hence, approaches that can be used for plant lipid discovery are also discussed.

Chenopodium quinoa (quinoa) is considered a superfood with its favourable nutrient composition and being gluten free. Quinoa has high tolerance to abiotic stresses, such as salinity, water deficit (drought) and cold. The tolerance mechanisms are yet to be elucidated. Quinoa has epidermal bladder cells (EBCs) that densely cover the shoot surface, particularly the younger parts of the plant. Here, we report on the EBC's primary and secondary metabolomes, as well as the lipidome in control conditions and in response to abiotic stresses. EBCs were isolated from plants after cold, heat, high-light, water deficit and salt treatments. We used untargeted gas chromatography-mass spectrometry (GC-MS) to analyse metabolites and untargeted and targeted liquid chromatography-MS (LC-MS) for lipids and secondary metabolite analyses. We identified 64 primary metabolites, including sugars, organic acids and amino acids, 19 secondary metabolites, including phenolic compounds, betanin and saponins and 240 lipids categorized in five groups including glycerolipids and phospholipids. We found only few changes in the metabolic composition of EBCs in response to abiotic stresses; these were metabolites related with heat, cold and high-light treatments but not salt stress. Na+ concentrations were low in EBCs with all treatments and approximately two orders of magnitude lower than K+ concentrations.

Chilling and frost conditions impose major yield restraints to wheat crops in Australia and other temperate climate regions. Unpredictability and variability of field frost events are major impediments for cold tolerance breeding. Metabolome and lipidome profiling were used to compare the cold response in spikes of cold-tolerant Young and sensitive variety Wyalkatchem at the young microspore (YM) stage of pollen development. We aimed to identify metabolite markers that can reliably distinguish cold-tolerant and sensitive wheat varieties for future cold-tolerance phenotyping applications. We scored changes in spike metabolites and lipids for both varieties during cold acclimation after initial and prolonged exposure to combined chilling and freezing cycles (1 and 4 days, respectively) using controlled environment conditions. The two contrasting wheat varieties showed qualitative and quantitative differences in primary metabolites involved in osmoprotection, but differences in lipid accumulation most distinctively separated the cold response of the two wheat lines. These results resemble what we previously observed in flag leaves of the same two wheat varieties. The fact that this response occurs in tissue types with very different functions indicates that chilling and freezing tolerance in these wheat lines is associated with re-modelling of membrane lipid composition to maintain membrane fluidity.

Locally synthesized sex steroids exert important paracrine actions in multiple tissues, including adipose and the heart. Quantifying these tissue steroids is essential in distinguishing how systemic and locally produced steroids exert their effects. Limited specificity and reproducibility of current methods (including ELISA) necessitate the development of new techniques. The aim of this study was to develop liquid chromatography tandem mass spectrometry/mass spectrometry (LC-MS/MS) techniques currently utilized in serum/plasma to quantify steroid levels in human/rodent pericardial adipose and myocardium.

Background and PurposeEndothelium‐derived vasoconstriction is a hallmark of vascular dysfunction in hypertension. In some cases, an overproduction of endothelium‐derived prostacyclin (PGI2) can cause contraction rather than relaxation. Relaxin is well known for its vasoprotective actions, but the possibility that this peptide could also reverse endothelium‐derived vasoconstriction has never been investigated. We tested the hypothesis that short‐term relaxin treatment mitigates endothelium‐derived vasoconstriction in spontaneously hypertensive rats (SHR).Experimental ApproachMale Wistar Kyoto rats (WKY) and SHR were subcutaneously infused with either vehicle (20 mmol·L−1 sodium acetate) or relaxin (13.3 μg·kg−1·hr−1) using osmotic minipumps for 3 days. Vascular reactivity to the endothelium‐dependent agonist ACh was assessed in vitro by wire myography. Quantitative PCR and LC‐MS were used to identify changes in gene expression of prostanoid pathways and PG production, respectively.Key ResultsRelaxin treatment ameliorated hypertension‐induced endothelial dysfunction by increasing NO‐dependent relaxation and reducing endothelium‐dependent contraction. Notably, short‐term relaxin treatment up‐regulated mesenteric PGI2 receptor (IP) expression, permitting PGI2–IP‐mediated vasorelaxation. In the aorta, reversal of contraction was accompanied by suppression of the hypertension‐induced increase in prostanoid‐producing enzymes and reduction in PGI2‐evoked contractions.Conclusions and ImplicationsRelaxin has region‐dependent vasoprotective actions in hypertension. Specifically, relaxin has distinct effects on endothelium‐derived contracting factors and their associated vasoconstrictor pathways in mesenteric arteries and the aorta. Taken together, these observations reveal the potential of relaxin as a new therapeutic agent for vascular disorders that are associated with endothelium‐derived vasoconstriction including hypertension.

Lipidomics is an emerging technology, which aims at the global characterization and quantification of lipids within biological matrices including biofluids, cells, whole organs and tissues. The changes in individual lipid molecular species in stress treated plant species and different cultivars can indicate the functions of genes affecting lipid metabolism or lipid signaling. Mass spectrometry-based lipid profiling has been used to track the changes of lipid levels and related metabolites in response to salinity stress. We have developed a comprehensive lipidomics platform for the identification and direct qualification and/or quantification of individual lipid species, including oxidized lipids, which enables a more systematic investigation of peroxidation of individual lipid species in barley roots under salinity stress. This new lipidomics approach has improved with an advantage of analyzing the composition of acyl chains at the molecular level, which facilitates to profile precisely the 18:3-containing diacyl-glycerophosphates and allowed individual comparison of lipids across varieties. Our findings revealed a general decrease in most of the galactolipids in plastid membranes, and an increase of glycerophospholipids and acylated steryl glycosides, which indicate that plastidial and extraplastidial membranes in barley roots ubiquitously tend to form a hexagonal II (HII) phase under salinity stress. In addition, salt-tolerant and salt-sensitive cultivars showed contrasting changes in the levels of oxidized membrane lipids. These results support the hypothesis that salt-induced oxidative damage to membrane lipids can be used as an indication of salt stress tolerance in barley.