Summary of Study ST003399

This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench, https://www.metabolomicsworkbench.org, where it has been assigned Project ID PR002105. The data can be accessed directly via it's Project DOI: 10.21228/M8HV52 This work is supported by NIH grant, U2C- DK119886.

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This study contains a large results data set and is not available in the mwTab file. It is only available for download via FTP as data file(s) here.

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Study IDST003399
Study TitleChemical Biology Meets Metabolomics: The Response of Barley Seedlings to 3,5-Dichloroanthranilic Acid, a Resistance Inducer
Study TypePlant metabolomics
Study SummaryAdvances in combinatorial synthesis and high-throughput screening methods have led to renewed interest in synthetic plant immune activators as well as priming agents. 3,5-Dichloroanthranilic acid (3,5-DCAA) is a derivative of anthranilic acid that have shown potency in activating defence mechanisms in Arabidopsis and barley plants. Chemical biology which is the interface of chemistry and biology can make use of metabolomics approaches and tools to better understand molecular mechanisms operating in complex biological systems. Aim: Here we report on the untargeted metabolomics profiling of barley seedlings treated with 3,5-DCAA to gain deeper insights into the mechanism of action of this resistance inducer. Methodology: Hydro-methanolic extracts from different time periods (12, 24 and 36 h post-treatment) were analysed on ultra-high performance liquid chromatography hyphenated with a high-resolution mass spectrometer. Both unsupervised and supervised chemometric methods were used to reveal hidden patterns and highlight metabolite variables associated with the treatment. Results: Based on the metabolites identified, both the phenylpropanoid and octadecanoid pathways appear to be main route activated by 3,5-DCAA. Different classes of responsive metabolites were annotated with favonoids, more especially flavones, the most dominant. Given the limited understanding of this inducer, this study offers a metabolomics analysis of the response triggered by its foliar application in barley. This additional insight could help make informed decision for the development of more effective strategies for crop protection and improvement, ultimately contributing to agricultural sustainability and resilience.
Institute
University of Johannesburg
DepartmentBiochemistry
LaboratoryPlant metabolomics
Last NameClaude Yasmine Hamany Djande
First NameClaude Yasmine
Address81A Fourth Avenue Westdene
Emailclaudehamany@gmail.com
Phone0814415123
Submit Date2024-07-05
Num Groups4
Raw Data AvailableYes
Raw Data File Type(s)raw(Waters)
Analysis Type DetailLC-MS
Release Date2024-09-03
Release Version1
Claude Yasmine Claude Yasmine Hamany Djande Claude Yasmine Claude Yasmine Hamany Djande
https://dx.doi.org/10.21228/M8HV52
ftp://www.metabolomicsworkbench.org/Studies/ application/zip

Select appropriate tab below to view additional metadata details:

Project:

Project ID:PR002105
Project DOI:doi: 10.21228/M8HV52
Project Title:Chemical Biology Meets Metabolomics: The Response of Barley Seedlings to 3,5-Dichloroanthranilic Acid, a Resistance Inducer
Project Type:Plant metabolomics
Project Summary:Advances in combinatorial synthesis and high-throughput screening methods have led to renewed interest in synthetic plant immune activators as well as priming agents. 3,5-Dichloroanthranilic acid (3,5-DCAA) is a derivative of anthranilic acid that have shown potency in activating defence mechanisms in Arabidopsis and barley plants. Chemical biology which is the interface of chemistry and biology can make use of metabolomics approaches and tools to better understand molecular mechanisms operating in complex biological systems. Aim: Here we report on the untargeted metabolomics profiling of barley seedlings treated with 3,5-DCAA to gain deeper insights into the mechanism of action of this resistance inducer. Methodology: Hydro-methanolic extracts from different time periods (12, 24 and 36 h post-treatment) were analysed on ultra-high performance liquid chromatography hyphenated with a high-resolution mass spectrometer. Both unsupervised and supervised chemometric methods were used to reveal hidden patterns and highlight metabolite variables associated with the treatment. Results: Based on the metabolites identified, both the phenylpropanoid and octadecanoid pathways appear to be main route activated by 3,5-DCAA. Different classes of responsive metabolites were annotated with favonoids, more especially flavones, the most dominant. Given the limited understanding of this inducer, this study offers a metabolomics analysis of the response triggered by its foliar application in barley. This additional insight could help make informed decision for the development of more effective strategies for crop protection and improvement, ultimately contributing to agricultural sustainability and resilience.
Institute:University of Johannesburg
Department:Biochemistry
Laboratory:Plant metabolomics
Last Name:Claude Yasmine Hamany Djande
First Name:Claude Yasmine
Address:81A Fourth Avenue Westdene
Email:claudehamany@gmail.com
Phone:0814415123

Subject:

Subject ID:SU003524
Subject Type:Plant
Subject Species:Hordeum vulgare
Taxonomy ID:4513
Age Or Age Range:3 weeks old
Gender:Not applicable

Factors:

Subject type: Plant; Subject species: Hordeum vulgare (Factor headings shown in green)

mb_sample_id local_sample_id Factor Sample source
SA371531120721ElimC12HC1bElimC12h Elim Control
SA371532120721ElimC12HA2bElimC12h Elim Control
SA371533120721ElimC12HC3bElimC12h Elim Control
SA371534120721ElimC12HC2bElimC12h Elim Control
SA371535120721ElimC12HA1bElimC12h Elim Control
SA371536120721ElimC12HB3bElimC12h Elim Control
SA371537120721ElimC12HB1bElimC12h Elim Control
SA371538120721ElimC12HA3bElimC12h Elim Control
SA371539120721ElimC12HB2bElimC12h Elim Control
SA371540120721ElimC24HA1bElimC24h Elim Control
SA371541120721ElimC24HA2bElimC24h Elim Control
SA371542120721ElimC24HA3bElimC24h Elim Control
SA371543120721ElimC24HB1bElimC24h Elim Control
SA371544120721ElimC24HB2bElimC24h Elim Control
SA371545120721ElimC24HB3bElimC24h Elim Control
SA371546120721ElimC24HC1bElimC24h Elim Control
SA371547120721ElimC24HC2bElimC24h Elim Control
SA371548120721ElimC24HC3bElimC24h Elim Control
SA371549120721ElimC36HC1bElimC36h Elim Control
SA371550120721ElimC36HC3bElimC36h Elim Control
SA371551120721ElimC36HC2bElimC36h Elim Control
SA371552120721ElimC36HA1bElimC36h Elim Control
SA371553120721ElimC36HB3bElimC36h Elim Control
SA371554120721ElimC36HB2bElimC36h Elim Control
SA371555120721ElimC36HB1bElimC36h Elim Control
SA371556120721ElimC36HA3bElimC36h Elim Control
SA371557120721ElimC36HA2bElimC36h Elim Control
SA371558120721ElimDCAA12HC3bElimDCAA12h Elim DCAA
SA371559120721ElimDCAA12HC1bElimDCAA12h Elim DCAA
SA371560120721ElimDCAA12HB3bElimDCAA12h Elim DCAA
SA371561120721ElimDCAA12HC2bElimDCAA12h Elim DCAA
SA371562120721ElimDCAA12HB1bElimDCAA12h Elim DCAA
SA371563120721ElimDCAA12HA3bElimDCAA12h Elim DCAA
SA371564120721ElimDCAA12HB2bElimDCAA12h Elim DCAA
SA371565120721ElimDCAA12HA2bElimDCAA12h Elim DCAA
SA371566120721ElimDCAA12HA1bElimDCAA12h Elim DCAA
SA371567120721ElimDCAA24HA2bElimDCAA24h Elim DCAA
SA371568120721ElimDCAA24HA3bElimDCAA24h Elim DCAA
SA371569120721ElimDCAA24HB1bElimDCAA24h Elim DCAA
SA371570120721ElimDCAA24HB2bElimDCAA24h Elim DCAA
SA371571120721ElimDCAA24HB3bElimDCAA24h Elim DCAA
SA371572120721ElimDCAA24HC1bElimDCAA24h Elim DCAA
SA371573120721ElimDCAA24HC2bElimDCAA24h Elim DCAA
SA371574120721ElimDCAA24HC3bElimDCAA24h Elim DCAA
SA371575120721ElimDCAA24HA1bElimDCAA24h Elim DCAA
SA371576120721ElimDCAA36HC1bElimDCAA36h Elim DCAA
SA371577120721ElimDCAA36HC3bElimDCAA36h Elim DCAA
SA371578120721ElimDCAA36HC2bElimDCAA36h Elim DCAA
SA371579120721ElimDCAA36HB1bElimDCAA36h Elim DCAA
SA371580120721ElimDCAA36HB3bElimDCAA36h Elim DCAA
SA371581120721ElimDCAA36HB2bElimDCAA36h Elim DCAA
SA371582120721ElimDCAA36HA2bElimDCAA36h Elim DCAA
SA371583120721ElimDCAA36HA1bElimDCAA36h Elim DCAA
SA371584120721ElimDCAA36HA3bElimDCAA36h Elim DCAA
SA371585120721QCAll1bQCAll QCAll
SA371586120721QCAll2bQCAll QCAll
SA371587120721QCAll3bQCAll QCAll
SA371588120721QCAll4bQCAll QCAll
SA371589120721QCAll5bQCAll QCAll
SA371590120721QCAll6bQCAll QCAll
SA371591120721QCAll7bQCAll QCAll
Showing results 1 to 61 of 61

Collection:

Collection ID:CO003517
Collection Summary:Seeds from the barley cultivar ‘Elim’ were provided by the South African Barley Breeding Institute (SABBI, Bredasdorp, Western Cape, South Africa). They were surfaced-sterilised with 70% ethanol and soaked in sterile water for 2 h prior to cultivation in soil (Germination mix, Culterra, Muldersdrift, South Africa) pasteurised at 70 °C. An average of 40 seeds were planted in each pot (three pots per condition or three biological replicate) measuring 8 cm depth and 12 cm in diameter. The watering of the plants was performed twice a week with water and a solution containing a water-soluble chemical fertiliser (Multisol ‘N’, Culterra, Muldersdrift, South Africa). Seedlings were kept in a regulated growth environment with a 12-hour light-dark cycle at 22 to 27°C until 16 d post-emergence or 21 d after planting, corresponding to physiological stage 13 according to the Zadocks growth and development scale (Zadoks et al. 1974). The priming inducer 3,5-dichloroanthranilic acid (3,5-DCAA) was purchased from Merck-Sigma-Aldrich, (Johannesburg, South Africa). 3,5-DCAA was dissolved in dimethylsulphoxide (DMSO, 1 μL.mL-1; BDH Chemicals, UK) and mixed with 0.05% wetting agent (Effekto, Pretoria, South Africa) in distilled water to obtain the desired concentrations. Approximately 6 mL (40 sprays) of 200 μM 3,5-DCAA was applied on the leaf tissue of the seedlings while the controls received only the DMSO solution. The leaf material was harvested at 12, 24 and 36 h post-treatment and snap-frozen in liquid nitrogen to quench metabolic activity. Samples were stored in -80 °C for later use. Metabolites were extracted as previously described (Hamany Djande et al., 2023b). Briefly, the leaf tissue was ground with liquid nitrogen, then 80% methanol was added and the mixture was homogenized. Subsequently, all hydromethanolic samples were centrifuged, and the supernatant was concentrated and further evaporated to complete dryness. The dried extracts were dissolved in 50% methanol, filtered, and prepared for LC-MS analysis.
Sample Type:Plant shoot tissue

Treatment:

Treatment ID:TR003533
Treatment Summary:3,5-DCAA was dissolved in dimethylsulphoxide (DMSO, 1 μL.mL-1; BDH Chemicals, UK) and mixed with 0.05% wetting agent (Effekto, Pretoria, South Africa) in distilled water to obtain the desired concentrations. Approximately 6 mL (40 sprays) of 200 μM 3,5-DCAA was applied on the leaf tissue of the seedlings while the controls received only the DMSO solution. The leaf material was harvested at 12, 24 and 36 h post-treatment and snap-frozen in liquid nitrogen to quench metabolic activity. Samples were stored in -80 °C for later use.
Treatment:synthetic inducer
Treatment Compound:3,5-DCAA
Treatment Dose:Approximately 6 mL (40 sprays) of 200 μM 3,5-DCAA
Treatment Dosevolume:6 mL
Plant Watering Regime:Twice a week
Plant Growth Stage:stage 13 according to the Zadocks growth and development scale (Zadoks et al. 1974)
Plant Storage:-80

Sample Preparation:

Sampleprep ID:SP003531
Sampleprep Summary:Metabolites were extracted as previously described (Hamany Djande et al., 2023b). Briefly, the leaf tissue was ground with liquid nitrogen, then 80% methanol was added and the mixture was homogenized. Subsequently, all hydromethanolic samples were centrifuged, and the supernatant was concentrated and further evaporated to complete dryness. The dried extracts were dissolved in 50% methanol, filtered, and prepared for LC-MS analysis.

Combined analysis:

Analysis ID AN005579
Analysis type MS
Chromatography type Reversed phase
Chromatography system Waters Acquity
Column Waters HSS T3 C18 (150 x 2.1mm x 1.8um)
MS Type ESI
MS instrument type QTOF
MS instrument name Waters Synapt G1
Ion Mode NEGATIVE
Units m/z_rt

Chromatography:

Chromatography ID:CH004239
Chromatography Summary:The analysis of the aqueous methanol extracts was performed with a Waters Acquity UHPLC coupled to a Waters SYNAPT G1 QTOF (quadrupole time-of-flight) high definition mass spectrometer system (Waters Corporation, Milford, MA, USA). The HSS T3 C18 column (150 mm x 2.1 mm x 1.8 µm, Waters Corporation) was thermostatted at 60 °C and used for the reverse phase chromatographic separation of extracts. The mobile phase consisted of mass spectrometry grade water and acetonitrile (Romil, SpS, Cambridge, UK) and formic acid (Sigma-Aldrich, Merck, Johannesburg, South Africa). Eluents A (water), and B (acetonitrile), both containing 0.1% formic acid were used for the concave gradient elution running at a flow rate of 0.4 mL min−1. The elution commenced with 5% B for the first min, and gradually increased to 95% B over 24 min. The chromatographic conditions were then adjusted to 10% A and 90% B, for 10 s, followed by 5% A and 95% B for 1 min 50 s before restoration to the initial conditions for column equilibration for 2 min. The injection volume was 2 µL and the total run time was 30 min. To account for analytical variability and to prevent measurement bias, each sample was analysed in triplicate. The sample order was randomised and blanks consisting of 50% methanol were injected to monitor the background noise, possible sample carry-over and solvent contamination. The stability of the LC-MS system was monitored by inserting quality control (QC) samples in the batches. Data acquisition involved three independent biological replicates, with each replicate analysed in triplicate, resulting in a total sample size of n = 9.
Instrument Name:Waters Acquity
Column Name:Waters HSS T3 C18 (150 x 2.1mm x 1.8um)
Column Temperature:60
Flow Gradient:The elution commenced with 5% B for the first min, and gradually increased to 95% B over 24 min. The chromatographic conditions were then adjusted to 10% A and 90% B, for 10 s, followed by 5% A and 95% B for 1 min 50 s before restoration to the initial conditions for column equilibration for 2 min. The injection volume was 2 µL and the total run time was 30 min
Flow Rate:0.4 mL/min
Solvent A:100% water; 0.1% formic acid
Solvent B:100% acetonitrile; 0.1% formic acid
Chromatography Type:Reversed phase

MS:

MS ID:MS005304
Analysis ID:AN005579
Instrument Name:Waters Synapt G1
Instrument Type:QTOF
MS Type:ESI
MS Comments:The high resolution, accurate mass TOF-MS analyser was used in a V-optics mode, with the centroid spectral data acquired in negative electrospray ionisation (ESI) modes. Operating parameters included masses ranging from 50 to 1200 Da and a scan time of 0.1 s; the capillary voltage set at 2.5 kV; sampling and extraction cone voltages at 40 V and 4.0 V, respectively. The desolvation and cone gas flows were set at 550 L h−1 and 50 L h−1, respectively, with nitrogen used as the nebulisation gas at a flow rate of 700 L h−1. A desolvation temperature of 450 °C and a fixed source temperature of 120 °C were used. Leucine encephalin ([M-H]− = 554.2615 and [M + H]+ = 556.2766) at a concentration of 50 pg mL−1, served as the reference mass calibrant, and was sampled every 15 sec to generate an average intensity of 350 counts per scan. This reference helped the processing software (MassLynx XSTM 4.1, Waters Corporation, Milford, MA, USA) to perform automatic correction of slight centroid mass deviations observed in the samples, ensuring precise mass measurements with typical mass accuracy ranging from 1 to 3 mDa. Both intact and fragmented data were aquired using an MSE method with collision energies ranging from 10 to 40 eV. The fragmentation data were employed for subsequent metabolite structural elucidation and annotation.
Ion Mode:NEGATIVE
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