Integration of targeted metabolome and transcript profiling of Pseudomonas syringae-triggered changes in defence-related phytochemicals in oat plants.

MAIN CONCLUSION: A gene-to-metabolite approach afforded new insights regarding defence mechanisms in oat plants that can be incorporated into plant breeding programmes for the selection of markers and genes related to disease resistance. Monitoring metabolite levels and changes therein can complement and corroborate transcriptome (mRNA) data on plant-pathogen interactions, thus revealing mechanisms involved in pathogen attack and host defence. A multi-omics approach thus adds new layers of information such as identifying metabolites with antimicrobial properties, elucidating metabolomic profiles of infected and non-infected plants, and reveals pathogenic requirements for infection and colonisation. In this study, two oat cultivars (Dunnart and SWK001) were inoculated with Pseudomonas syringae pathovars, pathogenic and non-pathogenic on oat. Following inoculation, metabolites were extracted with methanol from leaf tissues at 2, 4 and 6 days post-infection and analysed by multiple reaction monitoring (MRM) on a triple quadrupole mass spectrometer system. Relatedly, mRNA was isolated at the same time points, and the cDNA analysed by quantitative PCR (RT-qPCR) for expression levels of selected gene transcripts associated with avenanthramide (Avn) biosynthesis. The targeted amino acids, hydroxycinnamic acids and Avns were successfully quantified. Distinct cultivar-specific differences in the metabolite responses were observed in response to pathogenic and non-pathogenic strains. Trends in aromatic amino acids and hydroxycinnamic acids seem to indicate stronger activation and flux through these pathways in Dunnart as compared to SWK001. A positive correlation between hydroxycinnamoyl-CoA:hydroxyanthranilate N-hydroxycinnamoyl transferase (HHT) gene expression and the abundance of Avn A in both cultivars was documented. However, transcript profiling of selected genes involved in Avn synthesis did not reveal a clear pattern to distinguish between the tolerant and susceptible cultivars.

Avenanthramides, Distinctive Hydroxycinnamoyl Conjugates of Oat, Avena sativa L.: An Update on the Biosynthesis, Chemistry, and Bioactivities.

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.

Metabolomic Characterisation of Discriminatory Metabolites Involved in Halo Blight Disease in Oat Cultivars Caused by Pseudomonas syringae pv. coronafaciens.

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.

The presence of oxygenated lipids in plant defense in response to biotic stress: a metabolomics appraisal.

Recent lipid-based findings suggest more direct roles for fatty acids and their degradation products in inducing/modulating various aspects of plant defense, e.g. as signaling molecules following stress responses that may regulate plant innate immunity. The synthesis of oxylipins is a highly dynamic process and occurs in both a developmentally regulated mode and in response to abiotic and biotic stresses. This mini-review summarizes the occurrence of free - and oxygenated fatty acid derivatives in plants as part of an orchestrated metabolic defense against pathogen attack. Oxygenated C18 derived polyunsaturated fatty acids were identified by untargeted metabolomics studies of a number of different plant-microbe pathosystems and may serve as potential biomarkers of oxidative stress. Untargeted metabolomics in combination with targeted lipidomics, can uncover previously unrecognized aspects of lipid mobilization during plant defense.

Metabolomics for Biomarker Discovery: Key Signatory Metabolic Profiles for the Identification and Discrimination of Oat Cultivars.

The first step in crop introduction-or breeding programmes-requires cultivar identification and characterisation. Rapid identification methods would therefore greatly improve registration, breeding, seed, trade and inspection processes. Metabolomics has proven to be indispensable in interrogating cellular biochemistry and phenotyping. Furthermore, metabolic fingerprints are chemical maps that can provide detailed insights into the molecular composition of a biological system under consideration. Here, metabolomics was applied to unravel differential metabolic profiles of various oat (Avena sativa) cultivars (Magnifico, Dunnart, Pallinup, Overberg and SWK001) and to identify signatory biomarkers for cultivar identification. The respective cultivars were grown under controlled conditions up to the 3-week maturity stage, and leaves and roots were harvested for each cultivar. Metabolites were extracted using 80% methanol, and extracts were analysed on an ultra-high performance liquid chromatography (UHPLC) system coupled to a quadrupole time-of-flight (qTOF) high-definition mass spectrometer analytical platform. The generated data were processed and analysed using multivariate statistical methods. Principal component analysis (PCA) models were computed for both leaf and root data, with PCA score plots indicating cultivar-related clustering of the samples and pointing to underlying differential metabolic profiles of these cultivars. Further multivariate analyses were performed to profile differential signatory markers, which included carboxylic acids, amino acids, fatty acids, phenolic compounds (hydroxycinnamic and hydroxybenzoic acids, and associated derivatives) and flavonoids, among the respective cultivars. Based on the key signatory metabolic markers, the cultivars were successfully distinguished from one another in profiles derived from both leaves and roots. The study demonstrates that metabolomics can be used as a rapid phenotyping tool for cultivar differentiation.