Search Results (45)

Advances in combinatorial synthesis and high-throughput screening methods have led to renewed interest in synthetic plant immunity activators as well as priming agents. 3,5-Dichloroanthranilic acid (3,5-DCAA) is a derivative of anthranilic acid that has shown potency in activating defence mechanisms in Arabidopsis and barley. Chemical biology, which is the interface of chemistry and biology, can make use of metabolomic approaches and tools to better understand molecular mechanisms operating in complex biological systems. Here we report on the untargeted metabolomic profiling of barley seedlings treated with 3,5-DCAA to gain deeper insights into the mechanism of action of this resistance inducer. Histochemical analysis revealed the production of reactive oxygen species in the leaves upon 3,5-DCAA infiltration. Subsequently, methanolic extracts from different time periods (12, 24, and 36 h post-treatment) were analysed by ultra-high-performance liquid chromatography hyphenated to a high-resolution mass spectrometer. Both unsupervised and supervised chemometric methods were used to reveal hidden patterns and highlight metabolite variables associated with the treatment. Based on the metabolites identified, both the phenylpropanoid and octadecanoid pathways appear to be main routes activated by 3,5-DCAA. Different classes of responsive metabolites were annotated with flavonoids, more specifically flavones, which were the most dominant. Given the limited understanding of this inducer, this study offers a metabolomic analysis of the response triggered by its foliar application in barley. This additional insight could help make informed decisions for the development of more effective strategies for crop protection and improvement, ultimately contributing to crop resilience and agricultural sustainability. Full article

Specialized metabolites are produced via discrete metabolic pathways. These small molecules play significant roles in plant growth and development, as well as defense against environmental stresses. These include damping off or seedling blight at a post-emergence stage. Targeted metabolomics was followed to gain insights into metabolome changes characteristic of different developmental stages of sorghum seedlings. Metabolites were extracted from leaves at seven time points post-germination and analyzed using ultra-high performance liquid chromatography coupled to mass spectrometry. Multivariate statistical analysis combined with chemometric tools, such as principal component analysis, hierarchical clustering analysis, and orthogonal partial least squares–discriminant analysis, were applied for data exploration and to reduce data dimensionality as well as for the selection of potential discriminant biomarkers. Changes in metabolome patterns of the seedlings were analyzed in the early, middle, and late stages of growth (7, 14, and 29 days post-germination). The metabolite classes were amino acids, organic acids, lipids, cyanogenic glycosides, hormones, hydroxycinnamic acid derivatives, and flavonoids, with the latter representing the largest class of metabolites. In general, the metabolite content showed an increase with the progression of the plant growth stages. Most of the differential metabolites were derived from tryptophan and phenylalanine, which contribute to innate immune defenses as well as growth. Quantitative analysis identified a correlation of apigenin flavone derivatives with growth stage. Data-driven investigations of these metabolomes provided new insights into the developmental dynamics that occur in seedlings to limit post-germination mortality. Full article

Necrotrophic fungi affect a wide range of plants and cause significant crop losses. For the activation of multi-layered innate immune defences, plants can be primed or pre-conditioned to rapidly and more efficiently counteract this pathogen. Untargeted and targeted metabolomics analyses were applied to elucidate the biochemical processes involved in the response of 3,5-dichloroanthranilic acid (3,5-DCAA) primed barley plants to Pyrenophora teres f. teres (Ptt). A susceptible barley cultivar (‘Hessekwa’) at the third leaf growth stage was treated with 3,5-DCAA 24 h prior to infection using a Ptt conidia suspension. The infection was monitored over 2, 4, and 6 days post-inoculation. For untargeted studies, ultra-high performance liquid chromatography coupled with high-resolution mass spectrometry (UHPLC–MS) was used to analyse methanolic plant extracts. Acquired data were processed to generate the data matrices utilised in chemometric modelling and multi-dimensional data mining. For targeted studies, selected metabolites from the amino acids, phenolic acids, and alkaloids classes were quantified using multiple reaction monitoring (MRM) mass spectrometry. 3,5-DCAA was effective as a priming agent in delaying the onset and intensity of symptoms but could not prevent the progression of the disease. Unsupervised learning methods revealed clear differences between the sample extracts from the control plants and the infected plants. Both orthogonal projection to latent structure-discriminant analysis (OPLS-DA) and ‘shared and unique structures’ (SUS) plots allowed for the extraction of potential markers of the primed and naïve plant responses to Ptt. These include classes of organic acids, fatty acids, amino acids, phenolic acids, and derivatives and flavonoids. Among these, 5-oxo-proline and citric acid were notable as priming response-related metabolites. Metabolites from the tricarboxylic acid pathway were only discriminant in the primed plant infected with Ptt. Furthermore, the quantification of targeted metabolites revealed that hydroxycinnamic acids were significantly more prominent in the primed infected plants, especially at 2 d.p.i. Our research advances efforts to better understand regulated and reprogrammed metabolic responses that constitute defence priming in barley against Ptt. Full article

Designing innovative biological crop protection strategies to stimulate natural plant immunity is motivated by the growing need for eco-friendly alternatives to conventional biocidal agrochemicals. Salicylic acid (SA) and analogues are known chemical inducers of priming plant immunity against environmental stresses. The aim of the study was to study the metabolic reprogramming in barley plants following an application of three proposed dichlorinated inducers of acquired resistance. 3,5-Dichloroanthranilic acid, 2,6-dichloropyridine-4-carboxylic acid, and 3,5-dichlorosalicylic acid were applied to barley at the third leaf stage of development and harvested at 12, 24, and 36 h post-treatment. Metabolites were extracted using methanol for untargeted metabolomics analyses. Samples were analysed by ultra-high performance liquid chromatography coupled to high-definition mass spectrometry (UHPLC-HDMS). Chemometric methods and bioinformatics tools were used to mine and interpret the generated data. Alterations in the levels of both primary and secondary metabolites were observed. The accumulation of barley-specific metabolites, hordatines, and precursors was observed from 24 h post-treatment. The phenylpropanoid pathway, a marker of induced resistance, was identified among the key mechanisms activated by the treatment with the three inducers. No salicylic acid or SA derivatives were annotated as signatory biomarkers; instead, jasmonic acid precursors and derivatives were found as discriminatory metabolites across treatments. The study highlights differences and similarities in the metabolomes of barley after treatment with the three inducers and points to the triggering chemical changes associated with defence and resistance. This report is the first of its kind, and the knowledge acquired provides deeper insight into the role of dichlorinated small molecules as inducers of plant immunity and can be used in metabolomics-guided plant improvement programmes. Full article

Avenanthramides are a group of N-cinnamoylanthranilic acids (phenolic alkaloid compounds) that are produced in oat plants as phytoalexins, in response to pathogen attack and elicitation. The enzyme catalysing the cinnamamide-generating reaction is hydroxycinnamoyl-CoA: hydroxyanthranilate N-hydroxycinnamoyltransferase (HHT, a member of the super family of BAHD acyltransferases). HHT from oat appears to have a narrow range of substrate usage, with preferred use of 5-hydroxyanthranilic acid (and to a lesser extent, other hydroxylated and methoxylated derivatives) as acceptor molecules, but is able to use both substituted cinnamoyl-CoA and avenalumoyl-CoA thioesters as donor molecules. Avenanthramides thus combine carbon skeletons from both the stress-inducible shikimic acid and phenylpropanoid pathways. These features contribute to the chemical characteristics of avenanthramides as multifunctional plant defence compounds, as antimicrobial agents and anti-oxidants. Although avenanthramides are naturally and uniquely synthesised in oat plants, these molecules also exhibit medicinal and pharmaceutical uses important for human health, prompting research into utilisation of biotechnology to enhance agriculture and value-added production. Full article

Proteins play an essential regulatory role in the innate immune response of host plants following elicitation by either biotic or abiotic stresses. Isonitrosoacetophenone (INAP), an unusual oxime-containing stress metabolite, has been investigated as a chemical inducer of plant defence responses. Both transcriptomic and metabolomic studies of various INAP-treated plant systems have provided substantial insight into this compound’s defence-inducing and priming capabilities. To complement previous ‘omics’ work in this regard, a proteomic approach of time-dependent responses to INAP was followed. As such, Nicotiana tabacum (N. tabacum) cell suspensions were induced with INAP and changes monitored over a 24-h period. Protein isolation and proteome analysis at 0, 8, 16 and 24 h post-treatment were performed using two-dimensional electrophoresis followed by the gel-free eight-plex isobaric tags for relative and absolute quantitation (iTRAQ) based on liquid chromatography and mass spectrometry. Of the identified differentially abundant proteins, 125 were determined to be significant and further investigated. INAP treatment elicited changes to the proteome that affected proteins from a wide range of functional categories: defence, biosynthesis, transport, DNA and transcription, metabolism and energy, translation and signalling and response regulation. The possible roles of the differentially synthesised proteins in these functional classes are discussed. Results indicate up-regulated defence-related activity within the investigated time period, further highlighting a role for proteomic changes in priming as induced by INAP treatment. Full article

Essential oils are vital constituents of oil-bearing plants. However, their screening still demands harvesting of the plant for laboratory analysis. We report herein a simple, rapid and robust headspace bubble-in-drop microextraction screening technique (BID-SPME) requiring only small amounts of plant material. The optimised method uses 0.5 g of the crushed plant leaves sample obtained in a 2 mL capped chromatography vial, heated to 55 °C and sampled with 2 µL heptadecane in a Hamilton gastight syringe equilibrated for 15 min exposed to the headspace volume. The method was applied to three plants, Pinus radiata, Tagetes minuta and Artemisia afra, which are known for their essential oil content. The method was able to extract at least 80% of the oil constituents in such abundance that they could be easily annotated using the gas chromatography–mass spectrometry (GC–MS) mass spectral libraries. The major volatile organic compounds (VOCs) detected included tagetone, terpinen-4-ol, ocimenone, caryophyllene, dihydrotagetone, terpinolene and artemisia ketone, just to mention a few, at different concentrations in different plants. Importantly, these annotated VOCs were also reported in other studies in the same and even different plants, extracted using normal steam distillation and importantly those reported in the literature for different extraction techniques. Full article

Hyphozyma roseonigra is a dimorphic yeast used as a biocatalyst to convert sclareol, a plant diterpenoid to ambradiol. The latter is an intermediate in the synthesis of ambrafuran, a high-value chemical in the fragrance industry. Unfortunately, little is known about the underlying biochemistry of this microorganism. In this study, the integration of multi-platform-based metabolomics was used to better comprehend H. roseonigra from a biochemical perspective. The focus on metabolomic changes during growth and development was accomplished using untargeted LC–MS and NMR analyses. Cell suspensions were grown in batch culture over a 14-day period, and cells from the early-, log-, and stationary phases were harvested every second day using platform-compatible extraction procedures. Following chemometric analysis of LC–MS and NMR data acquired from both intra- and extracellular extracts, the identified discriminatory ions annotated from the endo- and exometabolomes (metabo-fingerprinting and metabo-footprinting) were found to fall predominantly in the primary metabolism class. Pathway mapping and feature-based network correlation analysis assisted in gaining insights into the active metabolic pathways during growth and development and did not flag terpene synthesis. This study provides novel insights into the basic metabolic capabilities of H. roseonigra and suggests that sclareol is metabolized as the detoxification of a hydrophobic xenobiotic compound. Full article

The rhizosphere microbiome is a major determinant of plant health. Plant-beneficial or plant growth-promoting rhizobacteria (PGPR) influence plant growth, plant development and adaptive responses, such as induced resistance/priming. These new eco-friendly choices have highlighted volatile organic compounds (biogenic VOCs) as a potentially inexpensive, effective and efficient substitute for the use of agrochemicals. Secreted bacterial VOCs are low molecular weight lipophilic compounds with a low boiling point and high vapor pressures. As such, they can act as short- or long-distance signals in the rhizosphere, affecting competing microorganisms and impacting plant health. In this study, secreted VOCs from four PGPR strains (Pseudomonas koreensis (N19), Ps. fluorescens (N04), Lysinibacillus sphaericus (T19) and Paenibacillus alvei (T22)) were profiled by solid-phase micro-extraction gas chromatography mass spectrometry (SPME–GC–MS) combined with a multivariate data analysis. Metabolomic profiling with chemometric analyses revealed novel data on the composition of the secreted VOC blends of the four PGPR strains. Of the 121 annotated metabolites, most are known as bioactives which are able to affect metabolism in plant hosts. These VOCs belong to the following classes: alcohols, aldehydes, ketones, alkanes, alkenes, acids, amines, salicylic acid derivatives, pyrazines, furans, sulfides and terpenoids. The results further demonstrated the presence of species-specific and strain-specific VOCs, characterized by either the absence or presence of specific VOCs in the different strains. These molecules could be further investigated as biomarkers for the classification of an organism as a PGPR and selection for agricultural use. Full article

Plants recognise bacterial microbe-associated molecular patterns (MAMPs) from the environment via plasma membrane (PM)-localised pattern recognition receptor(s) (PRRs). Lipopolysaccharides (LPSs) are known as MAMPs from gram-negative bacteria that are most likely recognised by PRRs and trigger defence responses in plants. The Arabidopsis PRR(s) and/or co-receptor(s) complex for LPS and the associated defence signalling remains elusive. As such, proteomic identification of LPS receptors and/or co-receptor complexes will help to elucidate the molecular mechanisms that underly LPS perception and defence signalling in plants. The Arabidopsis LPS-binding protein (LBP) and bactericidal/permeability-increasing protein (BPI)-related-2 (LBR2) have been shown to recognise LPS and trigger defence responses while brassinosteroid insensitive 1 (BRI1)-associated receptor kinase 1 (BAK1) acts as a co-receptor for several PRRs. In this study, Arabidopsis wild type (WT) and T-DNA knock out mutants (lbr2-2 and bak1-4) were treated with LPS chemotypes from Pseudomonas syringae pv. tomato DC3000 (Pst) and Xanthomonas campestris pv. campestris 8004 (Xcc) over a 24 h period. The PM-associated protein fractions were separated by liquid chromatography and analysed by tandem mass spectrometry (LC-MS/MS) followed by data analysis using Byonic^TM software. Using Gene Ontology (GO) for molecular function and biological processes, significant LPS-responsive proteins were grouped according to defence and stress response, perception and signalling, membrane transport and trafficking, metabolic processes and others. Venn diagrams demarcated the MAMP-responsive proteins that were common and distinct to the WT and mutant lines following treatment with the two LPS chemotypes, suggesting contributions from differential LPS sub-structural moieties and involvement of LBR2 and BAK1 in the LPS-induced MAMP-triggered immunity (MTI). Moreover, the identification of RLKs and RLPs that participate in other bacterial and fungal MAMP signalling proposes the involvement of more than one receptor and/or co-receptor for LPS perception as well as signalling in Arabidopsis defence responses. Full article

Plants perceive pathogenic threats from the environment that have evaded preformed barriers through pattern recognition receptors (PRRs) that recognise microbe-associated molecular patterns (MAMPs). The perception of and triggered defence to lipopolysaccharides (LPSs) as a MAMP is well-studied in mammals, but little is known in plants, including the PRR(s). Understanding LPS-induced secondary metabolites and perturbed metabolic pathways in Arabidopsis will be key to generating disease-resistant plants and improving global plant crop yield. Recently, Arabidopsis LPS-binding protein (LBP) and bactericidal/permeability-increasing protein (BPI)-related proteins (LBP/BPI related-1) and (LBP/BPI related-2) were shown to perceive LPS from Pseudomonas aeruginosa and trigger defence responses. In turn, brassinosteroid insensitive 1 (BRI1)-associated receptor kinase 1 (BAK1) is a well-established co-receptor for several defence-related PRRs in plants. Due to the lack of knowledge pertaining to LPS perception in plants and given the involvement of the afore-mentioned proteins in MAMPs recognition, in this study, Arabidopsis wild type (WT) and mutant (lbr2-2 and bak1-4) plants were pressure-infiltrated with LPSs purified from Pseudomonas syringae pv. tomato DC3000 (Pst) and Xanthomonas campestris pv. campestris 8004 (Xcc). Metabolites were extracted from the leaves at four time points over a 24 h period and analysed by UHPLC-MS, generating distinct metabolite profiles. Data analysed using unsupervised and supervised multivariate data analysis (MVDA) tools generated results that reflected time- and treatment-related variations after both LPS chemotypes treatments. Forty-five significant metabolites were putatively annotated and belong to the following groups: glucosinolates, hydroxycinnamic acid derivatives, flavonoids, lignans, lipids, oxylipins, arabidopsides and phytohormones, while metabolic pathway analysis (MetPA) showed enrichment of flavone and flavanol biosynthesis, phenylpropanoid biosynthesis, alpha-linolenic acid metabolism and glucosinolate biosynthesis. Distinct metabolite accumulations depended on the LPS chemotype and the genetic background of the lbr2-2 and bak1-4 mutants. This study highlights the role of LPSs in the reprogramming Arabidopsis metabolism into a defensive state, and the possible role of LBR and BAK1 proteins in LPSs perception and thus plant defence against pathogenic bacteria. Full article

In the process of enhancing crop potential, metabolomics offers a unique opportunity to biochemically describe plant metabolism and to elucidate metabolite profiles that govern specific phenotypic characteristics. In this study we report an untargeted metabolomic profiling of shoots and roots of barley seedlings performed to reveal the chemical makeup therein at an early growth stage. The study was conducted on five cultivars of barley: ‘Overture’, ‘Cristalia’, ‘Deveron’, ‘LE7′ and ‘Genie’. Seedlings were grown for 16 days post germination under identical controlled conditions, and methanolic extracts were analysed on an ultra-high performance liquid chromatography coupled to high-resolution mass spectrometry (UHPLC–HRMS) system. In addition, an unsupervised pattern identification technique, principal component analysis (PCA), was performed to process the generated multidimensional data. Following annotation of specific metabolites, several classes were revealed, among which phenolic acids represented the largest group in extracts from both shoot and root tissues. Interestingly, hordatines, barley-specific metabolites, were not found in the root tissue. In addition, metabolomic profiling revealed metabolites potentially associated with the plants’ natural protection system against potential pathogens. The study sheds light on the chemical composition of barley at a young developmental stage and the information gathered could be useful in plant research and biomarker-based breeding programs. Full article

The metabolome is the underlying biochemical layer of the phenotype and offers a functional readout of the cellular mechanisms involved in a biological system. Since metabolites are considered end-products of regulatory processes at a cellular level, their levels are considered the definitive response of the biological system to genetic or environmental variations. The metabolome thus serves as a metabolic fingerprint of the biochemical events that occur in a biological system under specific conditions. In this study, an untargeted metabolomics approach was applied to elucidate biochemical processes implicated in oat plant responses to Pseudomonas syringae pv. coronafaciens (Ps-c) infection, and to identify signatory markers related to defence responses and disease resistance against halo blight. Metabolic changes in two oat cultivars (“Dunnart” and “SWK001”) responding to Ps-c, were examined at the three-leaf growth stage and metabolome changes monitored over a four-day post-inoculation period. Hydromethanolic extracts were analysed using an ultra-high-performance liquid chromatography (UHPLC) system coupled to a high-definition mass spectrometer (MS) analytical platform. The acquired multi-dimensional data were processed using multivariate statistical analysis and chemometric modelling. The validated chemometric models indicated time- and cultivar-related metabolic changes, defining the host response to the bacterial inoculation. Further multivariate analyses of the data were performed to profile differential signatory markers, putatively associated with the type of launched defence response. These included amino acids, phenolics, phenolic amides, fatty acids, flavonoids, alkaloids, terpenoids, lipids, saponins and plant hormones. Based on the results, metabolic alterations involved in oat defence responses to Ps-c were elucidated and key signatory metabolic markers defining the defence metabolome were identified. The study thus contributes toward a more holistic understanding of the oat metabolism under biotic stress. Full article

Plant growth-promoting rhizobacteria (PGPR) are beneficial microorganisms colonising the rhizosphere. PGPR are involved in plant growth promotion and plant priming against biotic and abiotic stresses. Plant–microbe interactions occur through chemical communications in the rhizosphere and a tripartite interaction mechanism between plants, pathogenic microbes and plant-beneficial microbes has been defined. However, comprehensive information on the rhizosphere communications between plants and microbes, the tripartite interactions and the biochemical implications of these interactions on the plant metabolome is minimal and not yet widely available nor well understood. Furthermore, the mechanistic nature of PGPR effects on induced systemic resistance (ISR) and priming in plants at the molecular and metabolic levels is yet to be fully elucidated. As such, research investigating chemical communication in the rhizosphere is currently underway. Over the past decades, metabolomics approaches have been extensively used in describing the detailed metabolome of organisms and have allowed the understanding of metabolic reprogramming in plants due to tripartite interactions. Here, we review communication systems between plants and microorganisms in the rhizosphere that lead to plant growth stimulation and priming/induced resistance and the applications of metabolomics in understanding these complex tripartite interactions. Full article

Plants continuously produce essential metabolites that regulate their growth and development. The enrichment of specific metabolites determines plant interactions with the immediate environment, and some metabolites become critical in defence responses against biotic and abiotic stresses. Here, an untargeted UHPLC-qTOF-MS approach was employed to profile metabolites of wheat cultivars resistant or susceptible to the pathogen Puccinia striiformis f. sp. tritici (Pst) and Aluminium (Al³⁺) toxicity. Multivariate statistical analysis (MVDA) tools, viz. principal component analysis (PCA) and hierarchical cluster analysis (HiCA) were used to qualify the correlation between the identified metabolites and the designated traits. A total of 100 metabolites were identified from primary and secondary metabolisms, including phenolic compounds, such as flavonoid glycosides and hydroxycinnamic acid (HCA) derivatives, fatty acids, amino acids, and organic acids. All metabolites were significantly variable among the five wheat cultivars. The Pst susceptible cultivars demonstrated elevated concentrations of HCAs compared to their resistant counterparts. In contrast, ‘Koonap’ displayed higher levels of flavonoid glycosides, which could point to its resistant phenotype to Pst and Al³⁺ toxicity. The data provides an insight into the metabolomic profiles and thus the genetic background of Pst- and Al³⁺-resistant and susceptible wheat varieties. This study demonstrates the prospects of applied metabolomics for chemotaxonomic classification, phenotyping, and potential use in plant breeding and crop improvement. Full article