Abstract
Background: The ability of plants to respond to and interact with their surroundings is impressive. They have evolved extensive, unique and complex multi-layered processes that include recognition, long-distance signalling, the ability to restore damage, metabolic reprogramming in support of defence responses and metabolic memory. Metabolomics is a high-throughput study of the metabolome within a biological system at a specific time-point, in which the use of different chemometrics tools provides valuable insights for the analysis of metabolites and metabolic responses and understanding of the underlying biology. Metabolomics is essential for understanding the chemical ‘fingerprints’ of plants under specific conditions such as priming, an adaptive mechanism used by plants to increase their defensive capacity.
Hypothesis: Metabolomics tools can be used to chemo-profile and differentiate among barley cultivars before and after treatment with priming agents (that are functional analogues of salicylic acid), and infection with Pyerenophora teres f. teres (Ptt), a necrotrophic pathogen and the causal agent of the ‘net blotch net form’ disease.
Aims: To use metabolomics approaches to investigate the metabolic profiles of ten cultivars of barley: (rainfall winter cultivars: ‘Erica’, ‘Agulhas’, ‘S16’, ‘Elim’ and ‘Hessekwa’) and (irrigated summer cultivars: ‘Overture’, ‘Cristalia’ ‘Deveron’, ‘LE7’ and ‘Genie’); and to reveal defence-related metabolites after treatment with dichlorinated priming agents: 3,5-dichlorosalicylic acid (3,5-DCSA), 2,6-dichloropyridine-4-carboxylic acid (2,6-DCP-4-CA) and 3,5-dichloroanthranilic acid (3,5-DCAA), and infection of the naïve and primed barley plants with Ptt.
Research Methodology: Shoot and root tissues of barley cultivars were harvested 21 d after planting. Only shoot tissues were explored for inducer treatment (priming state) and infection study (post-challenged primed state). Hydro-methanolic extracts from each condition were analysed on ultra-high performance liquid chromatography hyphenated with a high-resolution mass spectrometer (UHPLC–HRMS). In addition, ‘multiple reaction monitoring’ (MRM) triple quadrupole (QqQ) MS was used for targeted analysis. As part of chemometric analyses, the unsupervised learning methods, principal component analyses (PCA), and hierarchical cluster analysis (HiCA) were used, and supervised learning methods included orthogonal partial least squares-discriminant analysis (PLS-DA) and orthogonal projection to latent structures discriminant analysis (OPLS-DA). Annotated metabolites were mapped to metabolic pathways to link the identified metabolites into a biological context.
Results and Discussion: For metabolite profiling and cultivar differentiation, phenolic acids were the most prominent class of metabolites identified in both shoot and root tissues. In this class, barley-specific metabolites, hordatines, were found exclusively in the shoots. Saponarin (a C-glycosylated isovitexin) was the only flavonoid metabolite in the roots. For the inducers treatment, both primary and secondary metabolism were altered. Here the accumulation of hordatines was observed. Among the potential biomarkers, jasmonic acid derivatives were annotated in all treatments but not salicylic acid and derivatives. Finally, in the pathogen infection study, a delay in infection symptoms of the primed plant was observed. This interval corresponded to a faster response of barley to the pathogen. Phenolic acids and derivatives, flavonoids, amino acids, organic acids, and fatty acids were the discriminant classes of annotated metabolites. 5-oxo-proline, citric acid, and a linolenic acid derivative were associated with the primed response to Ptt.
Conclusion: These studies revealed the metabolite composition of barley associated with different physiological states and contributed significant insights into their biological role in plant growth and development, priming, and defence against a necrotrophic pathogen. Such knowledge will provide more information in plant breeding selection to develop new cultivars.