Metabolomic Reconfiguration in Primed Barley (Hordeum vulgare) Plants in Response to Pyrenophora teres f. teres Infection.

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.

Metabolic Reprogramming of Barley in Response to Foliar Application of Dichlorinated Functional Analogues of Salicylic Acid as Priming Agents and Inducers of Plant Defence.

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.

Hordatines and Associated Precursors Dominate Metabolite Profiles of Barley (Hordeum vulgare L.) Seedlings: A Metabolomics Study of Five Cultivars.

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.

A Metabolomics Approach and Chemometric Tools for Differentiation of Barley Cultivars and Biomarker Discovery.

One of the ultimate goals of plant breeding is the development of new crop cultivars capable of withstanding increasing environmental stresses, to sustain the constantly growing population and economic demands. Investigating the chemical composition of the above and underground tissues of cultivars is crucial for the understanding of common and specific traits thereof. Using an untargeted metabolomics approach together with appropriate chemometrics tools, the differential metabolite profiles of leaf and root extracts from five cultivars of barley ('Erica', 'Elim', 'Hessekwa', 'S16' and 'Agulhas') were explored and potential signatory biomarkers were revealed. The study was conducted on seedlings grown for 21 days under identical controlled conditions. An ultra-high performance liquid chromatography coupled to high-resolution mass spectrometry (UHPLC-HRMS) was employed to analyse hydromethanolic leaf and root extracts of barley cultivars. Furthermore, unsupervised and supervised learning algorithms were applied to mine the generated data and to pinpoint cultivar-specific metabolites. Among all the classes of metabolites annotated, phenolic acids and derivatives formed the largest group and also represented the most discriminatory metabolites. In roots, saponarin, an important allelochemical differentially distributed across cultivars, was the only flavonoid annotated. The application of an untargeted metabolomics approach in phenotyping grain crops such as barley was demonstrated, and the metabolites responsible for differentiating between the selected cultivars were revealed. The study provides insights into the chemical architecture of barley, an agro-economically relevant cereal crop; and reiterates the importance of metabolomics tools in plant breeding practices for crop improvement.

Mass Spectrometric Approaches to Study the Metabolism of Jasmonates: Biotransformation of Exogenously Supplemented Methyl Jasmonate by Cell Suspension Cultures of Moringa oleifera.

Jasmonic acid (JA) and derivatives play a crucial role in plant adaptation to environmental stress. The exogenous application of methyl jasmonate (MeJA) results in activation of stress-related genes and subsequent production of secondary metabolites. This implies the biotransformation of the hormone into a more active form. In this study, Moringa oleifera cell suspension cultures were treated with 100 µM MeJA. Methanolic cell extracts were analyzed with reverse phase ultrahigh performance liquid chromatography (UHPLC) coupled to a quadrupole time-of-flight high definition mass spectrometer (QTOF-HDMS, with MSE data acquisition). Using a targeted approach for jasmonate profiling, extracted ion chromatograms were generated. MS fragmentation patterns were subsequently derived and molecular formulae calculated from the MS data of each ion. Furthermore, triple quadrupole (QqQ) MS, operated in selected reaction monitoring (SRM) mode, was employed to target masses of interest. In addition to MeJA and JA, three jasmonoyl-amino acids were annotated: jasmonoyl-valine (JA-Val), jasmonoyl-isoleucine/leucine (JA-Ile/Leu), and jasmonoyl-phenylalanine (JA-Phe), based on characteristic precursor and product ions. Furthermore, JA conjugated to a hexose was observed, as well as hydroxylated and carboxylated derivatives of JA-amino acid conjugates. The data point to active metabolism of the externally added MeJA by the M. oleifera cells through biotransformation and bioconversion reactions that can be investigated in depth using advanced mass spectrometric analyses.