Lipopolysaccharides from Ralstonia solanacearum induce a broad metabolomic response in Solanum lycopersicum.

Ralstonia solanacearum, one of the most destructive crop pathogens worldwide, causes bacterial wilt disease in a wide range of host plants. The major component of the outer membrane of Gram-negative bacteria, lipopolysaccharides (LPS), has been shown to function as elicitors of plant defense leading to the activation of signaling and defense pathways in several plant species. LPS from a R. solanacearum strain virulent on tomato (LPSR. sol.), were purified, chemically characterized, and structurally elucidated. The lipid A moiety consisted of tetra- to hexa-acylated bis-phosphorylated disaccharide backbone, also decorated by aminoarabinose residues in minor species, while the O-polysaccharide chain consisted of either linear tetrasaccharide or branched pentasaccharide repeating units containing α-L-rhamnose, N-acetyl-ß-D-glucosamine, and ß-L-xylose. These properties might be associated with the evasion of host surveillance, aiding the establishment of the infection. Using untargeted metabolomics, the effect of LPSR. sol. elicitation on the metabolome of Solanum lycopersicum leaves was investigated across three incubation time intervals with the application of UHPLC-MS for metabolic profiling. The results revealed the production of oxylipins, e.g., trihydroxy octadecenoic acid and trihydroxy octadecadienoic acid, as well as several hydroxycinnamic acid amide derivatives, e.g., coumaroyl tyramine and feruloyl tyramine, as phytochemicals that exhibit a positive correlation to LPSR. sol. treatment. Although the chemical properties of these metabolite classes have been studied, the functional roles of these compounds have not been fully elucidated. Overall, the results suggest that the features of the LPSR. sol. chemotype aid in limiting or attenuating the full deployment of small molecular host defenses and contribute to the understanding of the perturbation and reprogramming of host metabolism during biotic immune responses.

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.

Altered metabolomic states elicited by Flg22 and FlgII-28 in Solanum lycopersicum: intracellular perturbations and metabolite defenses.

BACKGROUND: Surveillance of potential pathogens is a key feature of plant innate immunity. For non-self-recognition plants rely on the perception of pathogen-derived molecules. Early post-perception events activate signaling cascades, leading to the synthesis of defense-related proteins and specialized metabolites, thereby providing a broad-spectrum antimicrobial coverage. This study was concerned with tracking changes in the tomato plant metabolome following perception of the flagellum-derived elicitors (Flg22 and FlgII-28). RESULTS: Following an untargeted metabolomics workflow, the metabolic profiles of a Solanum lycopersicum cultivar were monitored over a time range of 16-32 h post-treatment. Liquid chromatography was used to resolve the complex mixture of metabolites and mass spectrometry for the detection of differences associated with the elicitor treatments. Stringent data processing and multivariate statistical tools were applied to the complex dataset to extract relevant metabolite features associated with the elicitor treatments. Following perception of Flg22 and FlgII-28, both elicitors triggered an oxidative burst, albeit with different kinetic responses. Signatory biomarkers were annotated from diverse metabolite classes which included amino acid derivatives, lipid species, steroidal glycoalkaloids, hydroxybenzoic acids, hydroxycinnamic acids and derivatives, as well as flavonoids. CONCLUSIONS: An untargeted metabolomics approach adequately captured the subtle and nuanced perturbations associated with elicitor-linked plant defense responses. The shared and unique features characterizing the metabolite profiles suggest a divergence of signal transduction events following perception of Flg22 vs. FlgII-28, leading to a differential reorganization of downstream metabolic pathways.

Metabolomic Evaluation of Ralstonia solanacearum Cold Shock Protein Peptide (csp22)-Induced Responses in Solanum lycopersicum.

Ralstonia solanacearum, the causal agent of bacterial wilt, is one of the most destructive bacterial plant pathogens. This is linked to its evolutionary adaptation to evade host surveillance during the infection process since many of the pathogen's associated molecular patterns escape recognition. However, a 22-amino acid sequence of R. solanacearum-derived cold shock protein (csp22) was discovered to elicit an immune response in the Solanaceae. Using untargeted metabolomics, the effects of csp22-elicitation on the metabolome of Solanum lycopersicum leaves were investigated. Additionally, the study set out to discover trends that may suggest that csp22 inoculation bestows enhanced resistance on tomato against bacterial wilt. Results revealed the redirection of metabolism toward the phenylpropanoid pathway and sub-branches thereof. Compared to the host response with live bacteria, csp22 induced a subset of the discriminant metabolites, but also metabolites not induced in response to R. solanacearum. Here, a spectrum of hydroxycinnamic acids (especially ferulic acid), their conjugates and derivatives predominated as signatory biomarkers. From a metabolomics perspective, the results support claims that csp22 pre-treatment of tomato plants elicits increased resistance to R. solanacearum infection and contribute to knowledge on plant immune systems operation at an integrative level. The functional significance of these specialized compounds may thus support a heightened state of defense that can be applied to ward off attacking pathogens or toward priming of defense against future infections.

Hydroxycinnamate Amides: Intriguing Conjugates of Plant Protective Metabolites.

The syntheses of aromatic monoamines and aliphatic polyamines (PAs) are responsive to environmental stresses, with some modulating aspects of plant defense. Conjugation of amines to hydroxycinnamic acids (HCAs) generates HCA amides (HCAAs), with the conjugates possessing properties from both compounds. Conjugation may reduce the polarity of the resulting metabolite and assist in translocation, stability, and compartmentalization. Recent metabolomic insights identified HCAAs as biomarkers during plant-pathogen interactions, supporting a functional role in defense. The conjugates may contribute to regulation of the dynamic metabolic pool of hydroxycinnamates. This review highlights the occurrence of aromatic amines (AAs) and PAs in stress metabolism, conjugation to HCAs, and the roles of HCAAs during host defense, adding emphasis on their involvement in hydrogen peroxide (H2O2) production and cell-wall strengthening.

Metabolomic Profiling of the Host Response of Tomato (Solanum lycopersicum) Following Infection by Ralstonia solanacearum.

Tomato (Solanum lycopersicum) is an important dietary source of bioactive phytochemicals and active breeding programs constantly produce new cultivars possessing superior and desirable traits. The phytopathogenic Ralstonia solanacearum, the causal agent of bacterial wilt, is a highly destructive bacterial disease with a high economic impact on tomato production. This study followed an untargeted metabolomic approach involving four tomato cultivars and aimed at the identification of secondary metabolites involved in plant defense after infection with R. solanacearum. Liquid chromatography coupled to mass spectrometry (LC-MS) in combination with multivariate data analysis and chemometric modelling were utilized for the identification of discriminant secondary metabolites. The total of 81 statistically selected features were annotated belonging to the metabolite classes of amino acids, organic acids, fatty acids, various derivatives of cinnamic acid and benzoic acids, flavonoids and steroidal glycoalkaloids. The results indicate that the phenylpropanoid pathway, represented by flavonoids and hydroxycinnamic acids, is of prime importance in the tomato defense response. The hydroxycinnamic acids esters of quinic acid, hexoses and glucaric acids were identified as signatory biomarkers, as well as the hydroxycinnamic acid amides to polyamines and tyramine. Interestingly, the rapid and differential accumulation of putrescine, dopamine, and tyramine derivatives, along with the presence of a newly documented metabolite, feruloyl serotonin, were documented in the infected plants. Metabolite concentration variability in the different cultivar tissues point to cultivar-specific variation in the speed and manner of resource redistribution between the host tissues. These metabolic phenotypes provide insights into the differential metabolic signatures underlying the defense metabolism of the four cultivars, defining their defensive capabilities to R. solanacearum.

Comparative Metabolic Phenotyping of Tomato (Solanum lycopersicum) for the Identification of Metabolic Signatures in Cultivars Differing in Resistance to Ralstonia solanacearum.

Tomato (Solanum lycopersicum) is an important dietary source which contains numerous bioactive phytochemicals. Active breeding programs constantly produce new cultivars possessing superior and desirable traits. However, the underlying molecular signatures that functionally describe these traits are yet to be elucidated. Thus, in this study we used an untargeted metabolomic approach to describe differential metabolic profiles of four cultivars described as having high to intermediate resistance to Ralstonia solanacearum. Metabolites were methanol-extracted from leaves, stems and root tissues and analyzed by liquid chromatography coupled with high definition mass spectrometry. Multivariate data analysis revealed cultivar-related differential metabolic phenotypes. A total of 41 metabolites were statistically selected and annotated, consisting of amino acids, organic acids, lipids, derivatives of cinnamic acid and benzoic acids, flavonoids and steroidal glycoalkaloids which were especially prominent in the two highly resistant cultivars. Interestingly, the less resistant cultivars had various fatty acid derivatives in root extracts that contributed to the differentiated metabolic signatures. Moreover, the metabolic phenotype of the STAR9008 (8SC) cultivar with intermediate resistance, was characterized by derivatives of cinnamic acids and flavonoids but at lower levels compared to the resistant cultivars. The 8SC cultivar also exhibited a lack of hydroxybenzoic acid biomarkers, which may be attributed to its lower resistance. These metabolic phenotypes provide insights into the differential metabolic signatures underlying the metabolism of these four cultivars, defining their respective phenotypic traits such as their resistance, tolerance or susceptibility to Ralstonia solanacearum.