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1.
BMC Plant Biol ; 21(1): 429, 2021 Sep 21.
Artículo en Inglés | MEDLINE | ID: mdl-34548030

RESUMEN

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.


Asunto(s)
Resistencia a la Enfermedad/genética , Resistencia a la Enfermedad/inmunología , Interacciones Huésped-Patógeno/genética , Interacciones Huésped-Patógeno/inmunología , Pseudomonas syringae/patogenicidad , Solanum lycopersicum/genética , Solanum lycopersicum/metabolismo , Productos Agrícolas/genética , Productos Agrícolas/metabolismo , Productos Agrícolas/microbiología , Regulación de la Expresión Génica de las Plantas , Solanum lycopersicum/microbiología , Metabolómica
2.
Int J Mol Sci ; 20(16)2019 Aug 14.
Artículo en Inglés | MEDLINE | ID: mdl-31416118

RESUMEN

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.


Asunto(s)
Interacciones Huésped-Patógeno , Metaboloma , Metabolómica , Enfermedades de las Plantas/microbiología , Ralstonia solanacearum/fisiología , Solanum lycopersicum/metabolismo , Solanum lycopersicum/microbiología , Cromatografía Líquida de Alta Presión , Cromatografía Liquida , Espectrometría de Masas , Metabolómica/métodos , Fitoquímicos/química , Fitoquímicos/metabolismo
3.
Rapid Commun Mass Spectrom ; 32(2): 121-132, 2018 Jan 30.
Artículo en Inglés | MEDLINE | ID: mdl-28990281

RESUMEN

RATIONALE: Liquid chromatography coupled to mass spectrometry (LC/MS) is a dominant analytical platform in metabolomics, because of the high sensitivity and resolution, thus enabling large-scale coverage of metabolomes. Correspondingly, electrospray ionisation (ESI) is the favoured ionisation method in untargeted LC/MS metabolomics given the ability to produce large numbers of ions. In the workflow of LC/ESI-MS metabolomics, maximising the ionisation efficiency over a wide mass range is inevitably an essential and determining step, subsequently defining the extent of coverage of the metabolome under investigation. Thus in this study, electronic factors related to the functioning of the ESI source, namely the capillary and sample cone voltages, were explored to investigate the influence on the data acquired in metabolomic investigations. METHODS: Hydromethanolic samples from an untargeted study (sorghum plants responding dynamically to fungal infection) were analysed on a high-resolution/definition LC/ESI-MS system. Here the capillary and sample cone voltages of the ZSpray™ ESI source were varied between 1.5-3.0 kV and 10.0-40.0 V, respectively. The acquired data were processed with MarkerLynx™ software and analysed using central composite design response surface methodology and chemometric approaches (principal component analysis and orthogonal projection latent structures-discriminant analysis). RESULTS: The results evidently demonstrate that both capillary and sampling cone voltages not only significantly influence the recorded MS signals with regard to the number and abundance of features, but also the overall structure of the collected data. This consequently impacts on the information extracted from the data and thus affects coverage of the metabolome. CONCLUSIONS: The observations postulate in that, untargeted LC/MS metabolomics, 'what you see is what you ionise'. Although there is convergence of collected data under different ESI conditions, the nuances observed indicate that the exploration of different ion source settings could be the best trade-off in expanding and maximising the metabolome coverage in untargeted metabolomic experiments.

4.
Int J Mol Sci ; 19(9)2018 Aug 29.
Artículo en Inglés | MEDLINE | ID: mdl-30158424

RESUMEN

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.


Asunto(s)
Cromatografía Liquida/métodos , Espectrometría de Masas/métodos , Ralstonia solanacearum/metabolismo , Solanum lycopersicum/metabolismo , Regulación de la Expresión Génica de las Plantas/genética , Hojas de la Planta/metabolismo , Raíces de Plantas/metabolismo , Tallos de la Planta/metabolismo
5.
BMC Plant Biol ; 17(1): 227, 2017 Nov 29.
Artículo en Inglés | MEDLINE | ID: mdl-29187153

RESUMEN

BACKGROUND: Plants respond to various stress stimuli by activating an enhanced broad-spectrum defensive ability. The development of novel resistance inducers represents an attractive, alternative crop protection strategy. In this regard, hexanoic acid (Hxa, a chemical elicitor) and azelaic acid (Aza, a natural signaling compound) have been proposed as inducers of plant defense, by means of a priming mechanism. Here, we investigated both the mode of action and the complementarity of Aza and Hxa as priming agents in Nicotiana tabacum cells in support of enhanced defense. RESULTS: Metabolomic analyses identified signatory biomarkers involved in the establishment of a pre-conditioned state following Aza and Hxa treatment. Both inducers affected the metabolomes in a similar manner and generated common biomarkers: caffeoylputrescine glycoside, cis-5-caffeoylquinic acid, feruloylglycoside, feruloyl-3-methoxytyramine glycoside and feruloyl-3-methoxytyramine conjugate. Subsequently, quantitative real time-PCR was used to investigate the expression of inducible defense response genes: phenylalanine ammonia lyase, hydroxycinnamoyl CoA quinate transferase and hydroxycinnamoyl transferase to monitor activation of the early phenylpropanoid pathway and chlorogenic acids metabolism, while ethylene response element-binding protein, small sar1 GTPase, heat shock protein 90, RAR1, SGT1, non-expressor of PR genes 1 and thioredoxin were analyzed to report on signal transduction events. Pathogenesis-related protein 1a and defensin were quantified to investigate the activation of defenses regulated by salicylic acid and jasmonic acid respectively. The qPCR results revealed differential expression kinetics and, in general (except for NPR1, Thionin and PR1a), the relative gene expression ratios observed in the Hxa-treated cells were significantly greater than the expression observed in the cells treated with Aza. CONCLUSIONS: The results indicate that Aza and Hxa have a similar priming effect through activation of genes involved in the establishment of systemic acquired resistance, associated with enhanced synthesis of hydroxycinnamic acids and related conjugates.


Asunto(s)
Caproatos/farmacología , Ácidos Dicarboxílicos/farmacología , Nicotiana/efectos de los fármacos , Biomarcadores , Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Metaboloma/efectos de los fármacos , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Transducción de Señal , Nicotiana/genética , Nicotiana/inmunología , Nicotiana/metabolismo , Factores de Transcripción/metabolismo , Transcriptoma/efectos de los fármacos
6.
Molecules ; 22(8)2017 Jul 27.
Artículo en Inglés | MEDLINE | ID: mdl-28749445

RESUMEN

Vernonia fastigiata is a multi-purpose nutraceutical plant with interesting biological properties. However, very little is known about its phytochemical composition and, thus the need for its phytochemical characterization. In the current study, an environmentally friendly method, pressurized hot water extraction (PHWE), was used to extract metabolites from the leaves of V. fastigiata at various temperatures (50 °C, 100 °C, 150 °C and 200 °C). Ultra-high performance liquid chromatography-quadrupole time of flight mass spectrometry (UHPLC-qTOF-MS) analysis in combination with chemometric methods, particularly principal component analysis (PCA) and liquid/gas chromatography mass spectrometry (XCMS) cloud plots, were used to descriptively visualize the data and identify significant metabolites extracted at various temperatures. A total of 25 different metabolites, including hydroxycinnamic acid derivatives, clovamide, deoxy-clovamide and flavonoids, were noted for the first time in this plant. Overall, an increase in extraction temperature resulted in an increase in metabolite extraction during PHWE. This study is the first scientific report on the phytochemical composition of V. fastigiata, providing insight into the components of the chemo-diversity of this important plant.


Asunto(s)
Cromatografía Líquida de Alta Presión/métodos , Calor , Espectrometría de Masas/métodos , Metabolómica/métodos , Fitoquímicos/análisis , Presión , Vernonia/química , Ácidos Cumáricos/química , Glicosilación , Metaboloma , Fitoquímicos/química , Análisis de Componente Principal , Quercetina/química , Agua
7.
Rapid Commun Mass Spectrom ; 30(8): 1011-8, 2016 Apr 30.
Artículo en Inglés | MEDLINE | ID: mdl-27003038

RESUMEN

RATIONALE: Caffeoylquinic acid (CQA) derivatives are a group of structurally diverse phytochemicals that have attracted attention due to their many health benefits. The structural diversity of these molecules is due in part to the presence of regio- and geometrical isomerism. This structural diversity hampers the accurate annotation of these molecules in plant extracts. Mass spectrometry (MS) is successfully used to differentiate between the different regioisomers of the CQA derivatives; however, the accurate discrimination of the geometrical isomers of these molecules has proven to be an elusive task. METHODS: UV-irradiated methanolic solutions of diCQA were analyzed using an ultra-high-performance liquid chromatography/quadrupole time-of-flight mass spectrometry (UHPLC/QTOFMS) method in negative ionisation mode. An in-source collision-induced dissociation (ISCID) method was optimized by varying both the capillary and cone voltages to achieve differential fragmentation patterns between UV-generated geometrical isomers of the diCQAs during MS analyses. RESULTS: Changes in the capillary voltage did not cause a significant difference to the fragmentation patterns of the four geometrical isomers, while changes in the cone voltage resulted in significant differences in the fragmentation patterns. The results also show, for the first time, the preferential formation of alkali metal (Li(+), Na(+) and K(+)) adducts by the cis geometrical isomers of diCQAs, compared to their trans counterparts. CONCLUSIONS: Optimized QTOFMS-based methods may be used to differentiate the geometrical isomers of diCQAs. Finally, additives such as metal salts to induce adduct formation can be applied as an alternative method to differentiate closely related isomers which could have been difficult to differentiate under normal MS settings.


Asunto(s)
Cromatografía Líquida de Alta Presión/métodos , Espectrometría de Masas/métodos , Metales Alcalinos/química , Ácido Quínico/análogos & derivados , Isomerismo , Ácido Quínico/análisis , Ácido Quínico/química
8.
Rapid Commun Mass Spectrom ; 29(20): 1874-8, 2015 Oct 30.
Artículo en Inglés | MEDLINE | ID: mdl-26411508

RESUMEN

RATIONALE: Metabolomics is a qualitative and quantitative measurement of the metabolite content of any biological system under a given physiological status. Due to the chemically diverse nature of these samples, metabolite identification is a difficult task, and development of alternative approaches, such as those based on mass spectrometry (MS), aimed at proper metabolite identification is required. METHODS: Compliance constants, a direct measure of mechanical bond strength, were used for the first time to predict the MS fragmentation patterns of different regional isomers of a ubiquitous plant metabolite, caffeoylquinic acid (CQA). The compliance constant of an ester bond linking caffeic acid and a quinic acid molecule in CQA was calculated using density functional theory and Wilson's G-matrix formalism to distinguish the isomers. The predicted fragmentation patterns were compared with mass spectra obtained using negative ion electrospray ionization ultra-high-performance liquid chromatography quadrupole time-of-flight mass spectrometry (UHPLC/QTOFMS). RESULTS: Our in silico results were found to be in correlation with our UHPLC/QTOFMS results, suggesting a potential application of compliance constant algorithms for the rationalization of complex mass spectrometric data. The results also show that the different configurations in stereochemistry that exist between different regional isomers contribute to the underlying energy of the surrounding bonds and the fragmentation thereof. CONCLUSIONS: The results of our pilot study suggest that computational modelling can be applied for metabolite identification during metabolomic data mining and Natural Product research in general.


Asunto(s)
Moringa oleifera/química , Extractos Vegetales/química , Cromatografía Líquida de Alta Presión , Metabolómica , Moringa oleifera/metabolismo , Proyectos Piloto , Extractos Vegetales/metabolismo , Espectrometría de Masa por Ionización de Electrospray
9.
Food Microbiol ; 44: 180-4, 2014 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-25084661

RESUMEN

Isolation of filamentous species of two Aspergillum genera from compound feeds produced in South Africa, and subsequent extraction of their individual DNA in this study, presents a simple but rapid molecular procedure for high through-put analysis of the individual morphological forms. DNA was successfully isolated from the Aspergillus spp. from agar cultures by use of a commercial kit. Agarose gel electrophoresis fractionation of the fungi DNA, showed distinct bands. The DNA extracted by this procedure appears to be relatively pure with a ratio absorbance at 260 and 280 nm. However, the overall morphological and molecular data indicated that 67.5 and 51.1% of feed samples were found to be contaminated with Aspergillus flavus and Aspergillus parasiticus, respectively, with poultry feed having the highest contamination mean level of 5.7 × 105 CFU/g when compared to cattle (mean: 4.0 × 106 CFU/g), pig (mean: 2.7 × 104 CFU/g) and horse (1.0 × 102 CFU) feed. This technique presents a readily achievable, easy to use method in the extraction of filamentous fungal DNA and it's identification. Hence serves as an important tool towards molecular study of these organisms for routine analysis check in monitoring and improving compound feed quality against fungal contamination.


Asunto(s)
Alimentación Animal/microbiología , Aspergillus flavus/crecimiento & desarrollo , Aspergillus flavus/aislamiento & purificación , Aspergillus/crecimiento & desarrollo , Aspergillus/aislamiento & purificación , Alimentación Animal/análisis , Animales , Aspergillus/clasificación , Aspergillus/genética , Aspergillus flavus/clasificación , Aspergillus flavus/genética , Bovinos , Contaminación de Alimentos/análisis , Caballos , Técnicas de Tipificación Micológica , Aves de Corral , Sudáfrica , Porcinos
10.
Metabolites ; 14(2)2024 Feb 07.
Artículo en Inglés | MEDLINE | ID: mdl-38393004

RESUMEN

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.

11.
Molecules ; 18(4): 4267-81, 2013 Apr 11.
Artículo en Inglés | MEDLINE | ID: mdl-23579994

RESUMEN

Centella asiatica is an important source of biologically active pentacyclic triterpenoids. The enhancement of the biosynthesis of the centellosides by manipulation of associated metabolic pathways is receiving much attention. Jasmonates play critical roles in plant metabolism by up-regulating the expression of genes related to secondary metabolites. Here, we investigated the effect of methyl jasmonate (MeJa) in C. asiatica through targeted metabolomic profiling of asiaticoside and madecassoside as well as their aglycones, asiatic acid and madecassic acid. Cell suspensions were treated with 0.2 mM MeJa for 2, 4 and 6 days. Liquid chromatography coupled to mass spectrometry (LC-MS) was used to explore induced changes in metabolite profiles, both qualitatively and quantitatively. Principal component analysis (PCA)-derived scores plots revealed clusters of sample replicates for control and treated samples at 2, 4 and 6 days while loading plots aided in identifying signatory biomarkers (asiatic acid and madecassic acid, as well as asiaticoside and madecassoside) that clearly demonstrate the variability between samples. In addition to increased biosynthesis of the targeted centelloids, other differential changes in the intracellular metabolite profiles reflected the response of the C. asiatica cells to the MeJa-treatment as a reprogramming of the metabolome.


Asunto(s)
Acetatos/análisis , Centella/química , Ciclopentanos/análisis , Metabolómica/métodos , Oxilipinas/análisis , Plantas Medicinales/química , Triterpenos/química , Acetatos/química , Cromatografía Líquida de Alta Presión , Ciclopentanos/química , Cromatografía de Gases y Espectrometría de Masas , Oxilipinas/química , Triterpenos Pentacíclicos/análisis , Triterpenos Pentacíclicos/química , Extractos Vegetales , Triterpenos/análisis
12.
Metabolites ; 13(5)2023 May 17.
Artículo en Inglés | MEDLINE | ID: mdl-37233707

RESUMEN

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.

13.
Metabolites ; 13(9)2023 Sep 07.
Artículo en Inglés | MEDLINE | ID: mdl-37755277

RESUMEN

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.

14.
Front Mol Biosci ; 10: 1232233, 2023.
Artículo en Inglés | MEDLINE | ID: mdl-37635940

RESUMEN

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.

15.
Front Plant Sci ; 14: 1103413, 2023.
Artículo en Inglés | MEDLINE | ID: mdl-37123830

RESUMEN

Plant-microbe interactions are a phenomenal display of symbiotic/parasitic relationships between living organisms. Plant growth-promoting rhizobacteria (PGPR) are some of the most widely investigated plant-beneficial microbes due to their capabilities in stimulating plant growth and development and conferring protection to plants against biotic and abiotic stresses. As such, PGPR-mediated plant priming/induced systemic resistance (ISR) has become a hot topic among researchers, particularly with prospects of applications in sustainable agriculture. The current study applies untargeted ultra-high performance liquid chromatography-high-definition mass spectrometry (UHPLC-HDMS) to investigate PGPR-based metabolic reconfigurations in the metabolome of primed wheat plants against Puccinia striiformis f. sp. tricti (Pst). A seed bio-priming approach was adopted, where seeds were coated with two PGPR strains namely Bacillus subtilis and Paenibacillus alvei (T22) and grown under controlled conditions in a glasshouse. The plants were infected with Pst one-week post-germination, followed by weekly harvesting of leaf material. Subsequent metabolite extraction was carried out for analysis on a UHPLC-HDMS system for data acquisition. The data was chemometrically processed to reveal the underlying trends and data structures as well as potential signatory biomarkers for priming against Pst. Results showed notable metabolic reprogramming in primary and secondary metabolism, where the amino acid and organic acid content of primed-control, primed-challenged and non-primed-challenged plants were differentially reprogrammed. Similar trends were observed from the secondary metabolism, in which primed plants (particularly primed-challenged) showed an up-regulation of phenolic compounds (flavonoids, hydroxycinnamic acids-HCAs- and HCA amides) compared to the non-primed plants. The metabolomics-based semi-quantitative and qualitative assessment of the plant metabolomes revealed a time-dependent metabolic reprogramming in primed-challenged and primed-unchallenged plants, indicating the metabolic adaptations of the plants to stripe rust infection over time.

16.
Anal Bioanal Chem ; 404(2): 367-72, 2012 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-22699233

RESUMEN

Metabolomics entails identification and quantification of all metabolites within a biological system with a given physiological status; as such, it should be unbiased. A variety of techniques are used to measure the metabolite content of living systems, and results differ with the mode of data acquisition and output generation. LC-MS is one of many techniques that has been used to study the metabolomes of different organisms but, although used extensively, it does not provide a complete metabolic picture. Recent developments in technology, for example the introduction of UPLC-ESI-MS, have, however, seen LC-MS become the preferred technique for metabolomics. Here, we show that when MS settings are varied in UPLC-ESI-MS, different metabolite profiles result from the same sample. During use of a Synapt UPLC-high definition MS instrument, the collision energy was continually altered (3, 10, 20, and 30 eV) during MS acquisition. PCA and OPLS-DA analysis of the generated UPLC-MS data of metabolites extracted from elicited tobacco cells revealed different clustering and different distribution patterns. As expected, ion abundance decreases with increasing collision energy, but, more importantly, results in unique multivariate data patterns from the same samples. Our findings suggest that different collision energy settings should be investigated during MS data acquisition because these can contribute to coverage of a wider range of the metabolome by UPLC-ESI-MS and prevent biased results.


Asunto(s)
Espectrometría de Masas/métodos , Metabolómica , Células Cultivadas , Cromatografía Liquida , Nicotiana/citología , Nicotiana/metabolismo
17.
Molecules ; 17(2): 1698-715, 2012 Feb 09.
Artículo en Inglés | MEDLINE | ID: mdl-22322447

RESUMEN

Plants have the ability to continuously respond to microbial signals in their environment. One of these stimuli is a steroid from fungal membranes, ergosterol, which does not occur in plants, but acts as a pathogen-associated molecular pattern molecule to trigger defence mechanisms. Here we investigated the effect of ergosterol on the secondary metabolites in tobacco (Nicotiana tabacum) cells by profiling the induced sesquiterpenoids. Suspensions of tobacco cells were treated with different concentrations (0-1,000 nM) of ergosterol and incubated for different time periods (0-24 h). Metabolites were extracted with a selective dispersive liquid-liquid micro-extraction method. Thin layer chromatography was used as a screening method for identification of sesquiterpenoids in tobacco extracts. Liquid chromatography coupled to mass spectrometry was used for quantitative and qualitative analyses. The results showed that ergosterol triggered differential changes in the metabolome of tobacco cells, leading to variation in the biosynthesis of secondary metabolites. Metabolomic analysis through principal component analysis-scores plots revealed clusters of sample replicates for ergosterol treatments of 0, 50, 150, 300 and 1,000 nM and time-dependent variation at 0, 6, 12, 18 and 24 h. Five bicyclic sesquiterpenoid phytoalexins, capsidiol, lubimin, rishitin, solavetivone and phytuberin, were identified as being ergosterol-induced, contributing to the altered metabolome.


Asunto(s)
Ergosterol/farmacología , Nicotiana/efectos de los fármacos , Sesquiterpenos/metabolismo , Células Cultivadas , Cromatografía Liquida , Análisis de Componente Principal , Nicotiana/citología , Nicotiana/metabolismo
18.
Metabolites ; 12(5)2022 Apr 22.
Artículo en Inglés | MEDLINE | ID: mdl-35629883

RESUMEN

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.

19.
Metabolites ; 12(4)2022 Mar 31.
Artículo en Inglés | MEDLINE | ID: mdl-35448497

RESUMEN

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.

20.
Metabolites ; 12(3)2022 Mar 16.
Artículo en Inglés | MEDLINE | ID: mdl-35323691

RESUMEN

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.

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