{
"METABOLOMICS WORKBENCH":{"STUDY_ID":"ST003399","ANALYSIS_ID":"AN005579","PROJECT_ID":"PR002105","VERSION":"1","CREATED_ON":"August 7, 2024, 9:21 am"},

"PROJECT":{"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"},

"STUDY":{"STUDY_TITLE":"Chemical Biology Meets Metabolomics: The Response of Barley Seedlings to 3,5-Dichloroanthranilic Acid, a Resistance Inducer","STUDY_TYPE":"Plant metabolomics","STUDY_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","NUM_GROUPS":"4"},

"SUBJECT":{"SUBJECT_TYPE":"Plant","SUBJECT_SPECIES":"Hordeum vulgare","TAXONOMY_ID":"4513","AGE_OR_AGE_RANGE":"3 weeks old","GENDER":"Not applicable"},
"SUBJECT_SAMPLE_FACTORS":[
{
"Subject ID":"Elim",
"Sample ID":"120721ElimC12HA1b",
"Factors":{"Factor":"ElimC12h","Sample source":"Elim Control"},
"Additional sample data":{"RAW_FILE_NAME(Rwaw file name)":"120721ElimC12HA1b"}
},
{
"Subject ID":"Elim",
"Sample ID":"120721ElimC12HA2b",
"Factors":{"Factor":"ElimC12h","Sample source":"Elim Control"},
"Additional sample data":{"RAW_FILE_NAME(Rwaw file name)":"120721ElimC12HA2b"}
},
{
"Subject ID":"Elim",
"Sample ID":"120721ElimC12HA3b",
"Factors":{"Factor":"ElimC12h","Sample source":"Elim Control"},
"Additional sample data":{"RAW_FILE_NAME(Rwaw file name)":"120721ElimC12HA3b"}
},
{
"Subject ID":"Elim",
"Sample ID":"120721ElimC12HB1b",
"Factors":{"Factor":"ElimC12h","Sample source":"Elim Control"},
"Additional sample data":{"RAW_FILE_NAME(Rwaw file name)":"120721ElimC12HB1b"}
},
{
"Subject ID":"Elim",
"Sample ID":"120721ElimC12HB2b",
"Factors":{"Factor":"ElimC12h","Sample source":"Elim Control"},
"Additional sample data":{"RAW_FILE_NAME(Rwaw file name)":"120721ElimC12HB2b"}
},
{
"Subject ID":"Elim",
"Sample ID":"120721ElimC12HB3b",
"Factors":{"Factor":"ElimC12h","Sample source":"Elim Control"},
"Additional sample data":{"RAW_FILE_NAME(Rwaw file name)":"120721ElimC12HB3b"}
},
{
"Subject ID":"Elim",
"Sample ID":"120721ElimC12HC1b",
"Factors":{"Factor":"ElimC12h","Sample source":"Elim Control"},
"Additional sample data":{"RAW_FILE_NAME(Rwaw file name)":"120721ElimC12HC1b"}
},
{
"Subject ID":"Elim",
"Sample ID":"120721ElimC12HC2b",
"Factors":{"Factor":"ElimC12h","Sample source":"Elim Control"},
"Additional sample data":{"RAW_FILE_NAME(Rwaw file name)":"120721ElimC12HC2b"}
},
{
"Subject ID":"Elim",
"Sample ID":"120721ElimC12HC3b",
"Factors":{"Factor":"ElimC12h","Sample source":"Elim Control"},
"Additional sample data":{"RAW_FILE_NAME(Rwaw file name)":"120721ElimC12HC3b"}
},
{
"Subject ID":"Elim",
"Sample ID":"120721ElimC24HA1b",
"Factors":{"Factor":"ElimC24h","Sample source":"Elim Control"},
"Additional sample data":{"RAW_FILE_NAME(Rwaw file name)":"120721ElimC24HA1b"}
},
{
"Subject ID":"Elim",
"Sample ID":"120721ElimC24HA2b",
"Factors":{"Factor":"ElimC24h","Sample source":"Elim Control"},
"Additional sample data":{"RAW_FILE_NAME(Rwaw file name)":"120721ElimC24HA2b"}
},
{
"Subject ID":"Elim",
"Sample ID":"120721ElimC24HA3b",
"Factors":{"Factor":"ElimC24h","Sample source":"Elim Control"},
"Additional sample data":{"RAW_FILE_NAME(Rwaw file name)":"120721ElimC24HA3b"}
},
{
"Subject ID":"Elim",
"Sample ID":"120721ElimC24HB1b",
"Factors":{"Factor":"ElimC24h","Sample source":"Elim Control"},
"Additional sample data":{"RAW_FILE_NAME(Rwaw file name)":"120721ElimC24HB1b"}
},
{
"Subject ID":"Elim",
"Sample ID":"120721ElimC24HB2b",
"Factors":{"Factor":"ElimC24h","Sample source":"Elim Control"},
"Additional sample data":{"RAW_FILE_NAME(Rwaw file name)":"120721ElimC24HB2b"}
},
{
"Subject ID":"Elim",
"Sample ID":"120721ElimC24HB3b",
"Factors":{"Factor":"ElimC24h","Sample source":"Elim Control"},
"Additional sample data":{"RAW_FILE_NAME(Rwaw file name)":"120721ElimC24HB3b"}
},
{
"Subject ID":"Elim",
"Sample ID":"120721ElimC24HC1b",
"Factors":{"Factor":"ElimC24h","Sample source":"Elim Control"},
"Additional sample data":{"RAW_FILE_NAME(Rwaw file name)":"120721ElimC24HC1b"}
},
{
"Subject ID":"Elim",
"Sample ID":"120721ElimC24HC2b",
"Factors":{"Factor":"ElimC24h","Sample source":"Elim Control"},
"Additional sample data":{"RAW_FILE_NAME(Rwaw file name)":"120721ElimC24HC2b"}
},
{
"Subject ID":"Elim",
"Sample ID":"120721ElimC24HC3b",
"Factors":{"Factor":"ElimC24h","Sample source":"Elim Control"},
"Additional sample data":{"RAW_FILE_NAME(Rwaw file name)":"120721ElimC24HC3b"}
},
{
"Subject ID":"Elim",
"Sample ID":"120721ElimC36HA1b",
"Factors":{"Factor":"ElimC36h","Sample source":"Elim Control"},
"Additional sample data":{"RAW_FILE_NAME(Rwaw file name)":"120721ElimC36HA1b"}
},
{
"Subject ID":"Elim",
"Sample ID":"120721ElimC36HA2b",
"Factors":{"Factor":"ElimC36h","Sample source":"Elim Control"},
"Additional sample data":{"RAW_FILE_NAME(Rwaw file name)":"120721ElimC36HA2b"}
},
{
"Subject ID":"Elim",
"Sample ID":"120721ElimC36HA3b",
"Factors":{"Factor":"ElimC36h","Sample source":"Elim Control"},
"Additional sample data":{"RAW_FILE_NAME(Rwaw file name)":"120721ElimC36HA3b"}
},
{
"Subject ID":"Elim",
"Sample ID":"120721ElimC36HB1b",
"Factors":{"Factor":"ElimC36h","Sample source":"Elim Control"},
"Additional sample data":{"RAW_FILE_NAME(Rwaw file name)":"120721ElimC36HB1b"}
},
{
"Subject ID":"Elim",
"Sample ID":"120721ElimC36HB2b",
"Factors":{"Factor":"ElimC36h","Sample source":"Elim Control"},
"Additional sample data":{"RAW_FILE_NAME(Rwaw file name)":"120721ElimC36HB2b"}
},
{
"Subject ID":"Elim",
"Sample ID":"120721ElimC36HB3b",
"Factors":{"Factor":"ElimC36h","Sample source":"Elim Control"},
"Additional sample data":{"RAW_FILE_NAME(Rwaw file name)":"120721ElimC36HB3b"}
},
{
"Subject ID":"Elim",
"Sample ID":"120721ElimC36HC1b",
"Factors":{"Factor":"ElimC36h","Sample source":"Elim Control"},
"Additional sample data":{"RAW_FILE_NAME(Rwaw file name)":"120721ElimC36HC1b"}
},
{
"Subject ID":"Elim",
"Sample ID":"120721ElimC36HC2b",
"Factors":{"Factor":"ElimC36h","Sample source":"Elim Control"},
"Additional sample data":{"RAW_FILE_NAME(Rwaw file name)":"120721ElimC36HC2b"}
},
{
"Subject ID":"Elim",
"Sample ID":"120721ElimC36HC3b",
"Factors":{"Factor":"ElimC36h","Sample source":"Elim Control"},
"Additional sample data":{"RAW_FILE_NAME(Rwaw file name)":"120721ElimC36HC3b"}
},
{
"Subject ID":"Elim",
"Sample ID":"120721ElimDCAA12HA1b",
"Factors":{"Factor":"ElimDCAA12h","Sample source":"Elim DCAA"},
"Additional sample data":{"RAW_FILE_NAME(Rwaw file name)":"120721ElimDCAA12HA1b"}
},
{
"Subject ID":"Elim",
"Sample ID":"120721ElimDCAA12HA2b",
"Factors":{"Factor":"ElimDCAA12h","Sample source":"Elim DCAA"},
"Additional sample data":{"RAW_FILE_NAME(Rwaw file name)":"120721ElimDCAA12HA2b"}
},
{
"Subject ID":"Elim",
"Sample ID":"120721ElimDCAA12HA3b",
"Factors":{"Factor":"ElimDCAA12h","Sample source":"Elim DCAA"},
"Additional sample data":{"RAW_FILE_NAME(Rwaw file name)":"120721ElimDCAA12HA3b"}
},
{
"Subject ID":"Elim",
"Sample ID":"120721ElimDCAA12HB1b",
"Factors":{"Factor":"ElimDCAA12h","Sample source":"Elim DCAA"},
"Additional sample data":{"RAW_FILE_NAME(Rwaw file name)":"120721ElimDCAA12HB1b"}
},
{
"Subject ID":"Elim",
"Sample ID":"120721ElimDCAA12HB2b",
"Factors":{"Factor":"ElimDCAA12h","Sample source":"Elim DCAA"},
"Additional sample data":{"RAW_FILE_NAME(Rwaw file name)":"120721ElimDCAA12HB2b"}
},
{
"Subject ID":"Elim",
"Sample ID":"120721ElimDCAA12HB3b",
"Factors":{"Factor":"ElimDCAA12h","Sample source":"Elim DCAA"},
"Additional sample data":{"RAW_FILE_NAME(Rwaw file name)":"120721ElimDCAA12HB3b"}
},
{
"Subject ID":"Elim",
"Sample ID":"120721ElimDCAA12HC1b",
"Factors":{"Factor":"ElimDCAA12h","Sample source":"Elim DCAA"},
"Additional sample data":{"RAW_FILE_NAME(Rwaw file name)":"120721ElimDCAA12HC1b"}
},
{
"Subject ID":"Elim",
"Sample ID":"120721ElimDCAA12HC2b",
"Factors":{"Factor":"ElimDCAA12h","Sample source":"Elim DCAA"},
"Additional sample data":{"RAW_FILE_NAME(Rwaw file name)":"120721ElimDCAA12HC2b"}
},
{
"Subject ID":"Elim",
"Sample ID":"120721ElimDCAA12HC3b",
"Factors":{"Factor":"ElimDCAA12h","Sample source":"Elim DCAA"},
"Additional sample data":{"RAW_FILE_NAME(Rwaw file name)":"120721ElimDCAA12HC3b"}
},
{
"Subject ID":"Elim",
"Sample ID":"120721ElimDCAA24HA1b",
"Factors":{"Factor":"ElimDCAA24h","Sample source":"Elim DCAA"},
"Additional sample data":{"RAW_FILE_NAME(Rwaw file name)":"120721ElimDCAA24HA1b"}
},
{
"Subject ID":"Elim",
"Sample ID":"120721ElimDCAA24HA2b",
"Factors":{"Factor":"ElimDCAA24h","Sample source":"Elim DCAA"},
"Additional sample data":{"RAW_FILE_NAME(Rwaw file name)":"120721ElimDCAA24HA2b"}
},
{
"Subject ID":"Elim",
"Sample ID":"120721ElimDCAA24HA3b",
"Factors":{"Factor":"ElimDCAA24h","Sample source":"Elim DCAA"},
"Additional sample data":{"RAW_FILE_NAME(Rwaw file name)":"120721ElimDCAA24HA3b"}
},
{
"Subject ID":"Elim",
"Sample ID":"120721ElimDCAA24HB1b",
"Factors":{"Factor":"ElimDCAA24h","Sample source":"Elim DCAA"},
"Additional sample data":{"RAW_FILE_NAME(Rwaw file name)":"120721ElimDCAA24HB1b"}
},
{
"Subject ID":"Elim",
"Sample ID":"120721ElimDCAA24HB2b",
"Factors":{"Factor":"ElimDCAA24h","Sample source":"Elim DCAA"},
"Additional sample data":{"RAW_FILE_NAME(Rwaw file name)":"120721ElimDCAA24HB2b"}
},
{
"Subject ID":"Elim",
"Sample ID":"120721ElimDCAA24HB3b",
"Factors":{"Factor":"ElimDCAA24h","Sample source":"Elim DCAA"},
"Additional sample data":{"RAW_FILE_NAME(Rwaw file name)":"120721ElimDCAA24HB3b"}
},
{
"Subject ID":"Elim",
"Sample ID":"120721ElimDCAA24HC1b",
"Factors":{"Factor":"ElimDCAA24h","Sample source":"Elim DCAA"},
"Additional sample data":{"RAW_FILE_NAME(Rwaw file name)":"120721ElimDCAA24HC1b"}
},
{
"Subject ID":"Elim",
"Sample ID":"120721ElimDCAA24HC2b",
"Factors":{"Factor":"ElimDCAA24h","Sample source":"Elim DCAA"},
"Additional sample data":{"RAW_FILE_NAME(Rwaw file name)":"120721ElimDCAA24HC2b"}
},
{
"Subject ID":"Elim",
"Sample ID":"120721ElimDCAA24HC3b",
"Factors":{"Factor":"ElimDCAA24h","Sample source":"Elim DCAA"},
"Additional sample data":{"RAW_FILE_NAME(Rwaw file name)":"120721ElimDCAA24HC3b"}
},
{
"Subject ID":"Elim",
"Sample ID":"120721ElimDCAA36HA1b",
"Factors":{"Factor":"ElimDCAA36h","Sample source":"Elim DCAA"},
"Additional sample data":{"RAW_FILE_NAME(Rwaw file name)":"120721ElimDCAA36HA1b"}
},
{
"Subject ID":"Elim",
"Sample ID":"120721ElimDCAA36HA2b",
"Factors":{"Factor":"ElimDCAA36h","Sample source":"Elim DCAA"},
"Additional sample data":{"RAW_FILE_NAME(Rwaw file name)":"120721ElimDCAA36HA2b"}
},
{
"Subject ID":"Elim",
"Sample ID":"120721ElimDCAA36HA3b",
"Factors":{"Factor":"ElimDCAA36h","Sample source":"Elim DCAA"},
"Additional sample data":{"RAW_FILE_NAME(Rwaw file name)":"120721ElimDCAA36HA3b"}
},
{
"Subject ID":"Elim",
"Sample ID":"120721ElimDCAA36HB1b",
"Factors":{"Factor":"ElimDCAA36h","Sample source":"Elim DCAA"},
"Additional sample data":{"RAW_FILE_NAME(Rwaw file name)":"120721ElimDCAA36HB1b"}
},
{
"Subject ID":"Elim",
"Sample ID":"120721ElimDCAA36HB2b",
"Factors":{"Factor":"ElimDCAA36h","Sample source":"Elim DCAA"},
"Additional sample data":{"RAW_FILE_NAME(Rwaw file name)":"120721ElimDCAA36HB2b"}
},
{
"Subject ID":"Elim",
"Sample ID":"120721ElimDCAA36HB3b",
"Factors":{"Factor":"ElimDCAA36h","Sample source":"Elim DCAA"},
"Additional sample data":{"RAW_FILE_NAME(Rwaw file name)":"120721ElimDCAA36HB3b"}
},
{
"Subject ID":"Elim",
"Sample ID":"120721ElimDCAA36HC1b",
"Factors":{"Factor":"ElimDCAA36h","Sample source":"Elim DCAA"},
"Additional sample data":{"RAW_FILE_NAME(Rwaw file name)":"120721ElimDCAA36HC1b"}
},
{
"Subject ID":"Elim",
"Sample ID":"120721ElimDCAA36HC2b",
"Factors":{"Factor":"ElimDCAA36h","Sample source":"Elim DCAA"},
"Additional sample data":{"RAW_FILE_NAME(Rwaw file name)":"120721ElimDCAA36HC2b"}
},
{
"Subject ID":"Elim",
"Sample ID":"120721ElimDCAA36HC3b",
"Factors":{"Factor":"ElimDCAA36h","Sample source":"Elim DCAA"},
"Additional sample data":{"RAW_FILE_NAME(Rwaw file name)":"120721ElimDCAA36HC3b"}
},
{
"Subject ID":"Elim",
"Sample ID":"120721QCAll1b",
"Factors":{"Factor":"QCAll","Sample source":"QCAll"},
"Additional sample data":{"RAW_FILE_NAME(Rwaw file name)":"120721QCAll1b"}
},
{
"Subject ID":"Elim",
"Sample ID":"120721QCAll2b",
"Factors":{"Factor":"QCAll","Sample source":"QCAll"},
"Additional sample data":{"RAW_FILE_NAME(Rwaw file name)":"120721QCAll2b"}
},
{
"Subject ID":"Elim",
"Sample ID":"120721QCAll3b",
"Factors":{"Factor":"QCAll","Sample source":"QCAll"},
"Additional sample data":{"RAW_FILE_NAME(Rwaw file name)":"120721QCAll3b"}
},
{
"Subject ID":"Elim",
"Sample ID":"120721QCAll4b",
"Factors":{"Factor":"QCAll","Sample source":"QCAll"},
"Additional sample data":{"RAW_FILE_NAME(Rwaw file name)":"120721QCAll4b"}
},
{
"Subject ID":"Elim",
"Sample ID":"120721QCAll5b",
"Factors":{"Factor":"QCAll","Sample source":"QCAll"},
"Additional sample data":{"RAW_FILE_NAME(Rwaw file name)":"120721QCAll5b"}
},
{
"Subject ID":"Elim",
"Sample ID":"120721QCAll6b",
"Factors":{"Factor":"QCAll","Sample source":"QCAll"},
"Additional sample data":{"RAW_FILE_NAME(Rwaw file name)":"120721QCAll6b"}
},
{
"Subject ID":"Elim",
"Sample ID":"120721QCAll7b",
"Factors":{"Factor":"QCAll","Sample source":"QCAll"},
"Additional sample data":{"RAW_FILE_NAME(Rwaw file name)":"120721QCAll7b"}
}
],
"COLLECTION":{"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_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"},

"SAMPLEPREP":{"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."},

"CHROMATOGRAPHY":{"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.","CHROMATOGRAPHY_TYPE":"Reversed phase","INSTRUMENT_NAME":"Waters Acquity","COLUMN_NAME":"Waters HSS T3 C18 (150 x 2.1mm x 1.8um)","SOLVENT_A":"100% water; 0.1% formic acid","SOLVENT_B":"100% acetonitrile; 0.1% formic acid","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","COLUMN_TEMPERATURE":"N/A"},

"ANALYSIS":{"ANALYSIS_TYPE":"MS"},

"MS":{"INSTRUMENT_NAME":"Waters SYNAPT G1 QTOF","INSTRUMENT_TYPE":"QTOF","MS_TYPE":"ESI","ION_MODE":"NEGATIVE","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.","MS_RESULTS_FILE":"ST003399_AN005579_Results.txt UNITS:m/z_rt Has m/z:Yes Has RT:Yes RT units:Minutes"}

}