#METABOLOMICS WORKBENCH Claude19_20240705_030929 DATATRACK_ID:4982 STUDY_ID:ST003399 ANALYSIS_ID:AN005579 PROJECT_ID:PR002105
VERSION             	1
CREATED_ON             	August 7, 2024, 9:21 am
#PROJECT
PR:PROJECT_TITLE                 	Chemical Biology Meets Metabolomics: The Response of Barley Seedlings to
PR:PROJECT_TITLE                 	3,5-Dichloroanthranilic Acid, a Resistance Inducer
PR:PROJECT_TYPE                  	Plant metabolomics
PR:PROJECT_SUMMARY               	Advances in combinatorial synthesis and high-throughput screening methods have
PR:PROJECT_SUMMARY               	led to renewed interest in synthetic plant immune activators as well as priming
PR:PROJECT_SUMMARY               	agents. 3,5-Dichloroanthranilic acid (3,5-DCAA) is a derivative of anthranilic
PR:PROJECT_SUMMARY               	acid that have shown potency in activating defence mechanisms in Arabidopsis and
PR:PROJECT_SUMMARY               	barley plants. Chemical biology which is the interface of chemistry and biology
PR:PROJECT_SUMMARY               	can make use of metabolomics approaches and tools to better understand molecular
PR:PROJECT_SUMMARY               	mechanisms operating in complex biological systems. Aim: Here we report on the
PR:PROJECT_SUMMARY               	untargeted metabolomics profiling of barley seedlings treated with 3,5-DCAA to
PR:PROJECT_SUMMARY               	gain deeper insights into the mechanism of action of this resistance inducer.
PR:PROJECT_SUMMARY               	Methodology: Hydro-methanolic extracts from different time periods (12, 24 and
PR:PROJECT_SUMMARY               	36 h post-treatment) were analysed on ultra-high performance liquid
PR:PROJECT_SUMMARY               	chromatography hyphenated with a high-resolution mass spectrometer. Both
PR:PROJECT_SUMMARY               	unsupervised and supervised chemometric methods were used to reveal hidden
PR:PROJECT_SUMMARY               	patterns and highlight metabolite variables associated with the treatment.
PR:PROJECT_SUMMARY               	Results: Based on the metabolites identified, both the phenylpropanoid and
PR:PROJECT_SUMMARY               	octadecanoid pathways appear to be main route activated by 3,5-DCAA. Different
PR:PROJECT_SUMMARY               	classes of responsive metabolites were annotated with favonoids, more especially
PR:PROJECT_SUMMARY               	flavones, the most dominant. Given the limited understanding of this inducer,
PR:PROJECT_SUMMARY               	this study offers a metabolomics analysis of the response triggered by its
PR:PROJECT_SUMMARY               	foliar application in barley. This additional insight could help make informed
PR:PROJECT_SUMMARY               	decision for the development of more effective strategies for crop protection
PR:PROJECT_SUMMARY               	and improvement, ultimately contributing to agricultural sustainability and
PR:PROJECT_SUMMARY               	resilience.
PR:INSTITUTE                     	University of Johannesburg
PR:DEPARTMENT                    	Biochemistry
PR:LABORATORY                    	Plant metabolomics
PR:LAST_NAME                     	Claude Yasmine Hamany Djande
PR:FIRST_NAME                    	Claude Yasmine
PR:ADDRESS                       	81A Fourth Avenue Westdene
PR:EMAIL                         	claudehamany@gmail.com
PR:PHONE                         	0814415123
#STUDY
ST:STUDY_TITLE                   	Chemical Biology Meets Metabolomics: The Response of Barley Seedlings to
ST:STUDY_TITLE                   	3,5-Dichloroanthranilic Acid, a Resistance Inducer
ST:STUDY_TYPE                    	Plant metabolomics
ST:STUDY_SUMMARY                 	Advances in combinatorial synthesis and high-throughput screening methods have
ST:STUDY_SUMMARY                 	led to renewed interest in synthetic plant immune activators as well as priming
ST:STUDY_SUMMARY                 	agents. 3,5-Dichloroanthranilic acid (3,5-DCAA) is a derivative of anthranilic
ST:STUDY_SUMMARY                 	acid that have shown potency in activating defence mechanisms in Arabidopsis and
ST:STUDY_SUMMARY                 	barley plants. Chemical biology which is the interface of chemistry and biology
ST:STUDY_SUMMARY                 	can make use of metabolomics approaches and tools to better understand molecular
ST:STUDY_SUMMARY                 	mechanisms operating in complex biological systems. Aim: Here we report on the
ST:STUDY_SUMMARY                 	untargeted metabolomics profiling of barley seedlings treated with 3,5-DCAA to
ST:STUDY_SUMMARY                 	gain deeper insights into the mechanism of action of this resistance inducer.
ST:STUDY_SUMMARY                 	Methodology: Hydro-methanolic extracts from different time periods (12, 24 and
ST:STUDY_SUMMARY                 	36 h post-treatment) were analysed on ultra-high performance liquid
ST:STUDY_SUMMARY                 	chromatography hyphenated with a high-resolution mass spectrometer. Both
ST:STUDY_SUMMARY                 	unsupervised and supervised chemometric methods were used to reveal hidden
ST:STUDY_SUMMARY                 	patterns and highlight metabolite variables associated with the treatment.
ST:STUDY_SUMMARY                 	Results: Based on the metabolites identified, both the phenylpropanoid and
ST:STUDY_SUMMARY                 	octadecanoid pathways appear to be main route activated by 3,5-DCAA. Different
ST:STUDY_SUMMARY                 	classes of responsive metabolites were annotated with favonoids, more especially
ST:STUDY_SUMMARY                 	flavones, the most dominant. Given the limited understanding of this inducer,
ST:STUDY_SUMMARY                 	this study offers a metabolomics analysis of the response triggered by its
ST:STUDY_SUMMARY                 	foliar application in barley. This additional insight could help make informed
ST:STUDY_SUMMARY                 	decision for the development of more effective strategies for crop protection
ST:STUDY_SUMMARY                 	and improvement, ultimately contributing to agricultural sustainability and
ST:STUDY_SUMMARY                 	resilience.
ST:INSTITUTE                     	University of Johannesburg
ST:DEPARTMENT                    	Biochemistry
ST:LABORATORY                    	Plant metabolomics
ST:LAST_NAME                     	Claude Yasmine Hamany Djande
ST:FIRST_NAME                    	Claude Yasmine
ST:ADDRESS                       	81A Fourth Avenue Westdene
ST:EMAIL                         	claudehamany@gmail.com
ST:PHONE                         	0814415123
ST:NUM_GROUPS                    	4
#SUBJECT
SU:SUBJECT_TYPE                  	Plant
SU:SUBJECT_SPECIES               	Hordeum vulgare
SU:TAXONOMY_ID                   	4513
SU:AGE_OR_AGE_RANGE              	3 weeks old
SU:GENDER                        	Not applicable
#SUBJECT_SAMPLE_FACTORS:         	SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Raw file names and additional sample data
SUBJECT_SAMPLE_FACTORS           	Elim	120721ElimC12HA1b	Factor:ElimC12h | Sample source:Elim Control	RAW_FILE_NAME(Rwaw file name)=120721ElimC12HA1b
SUBJECT_SAMPLE_FACTORS           	Elim	120721ElimC12HA2b	Factor:ElimC12h | Sample source:Elim Control	RAW_FILE_NAME(Rwaw file name)=120721ElimC12HA2b
SUBJECT_SAMPLE_FACTORS           	Elim	120721ElimC12HA3b	Factor:ElimC12h | Sample source:Elim Control	RAW_FILE_NAME(Rwaw file name)=120721ElimC12HA3b
SUBJECT_SAMPLE_FACTORS           	Elim	120721ElimC12HB1b	Factor:ElimC12h | Sample source:Elim Control	RAW_FILE_NAME(Rwaw file name)=120721ElimC12HB1b
SUBJECT_SAMPLE_FACTORS           	Elim	120721ElimC12HB2b	Factor:ElimC12h | Sample source:Elim Control	RAW_FILE_NAME(Rwaw file name)=120721ElimC12HB2b
SUBJECT_SAMPLE_FACTORS           	Elim	120721ElimC12HB3b	Factor:ElimC12h | Sample source:Elim Control	RAW_FILE_NAME(Rwaw file name)=120721ElimC12HB3b
SUBJECT_SAMPLE_FACTORS           	Elim	120721ElimC12HC1b	Factor:ElimC12h | Sample source:Elim Control	RAW_FILE_NAME(Rwaw file name)=120721ElimC12HC1b
SUBJECT_SAMPLE_FACTORS           	Elim	120721ElimC12HC2b	Factor:ElimC12h | Sample source:Elim Control	RAW_FILE_NAME(Rwaw file name)=120721ElimC12HC2b
SUBJECT_SAMPLE_FACTORS           	Elim	120721ElimC12HC3b	Factor:ElimC12h | Sample source:Elim Control	RAW_FILE_NAME(Rwaw file name)=120721ElimC12HC3b
SUBJECT_SAMPLE_FACTORS           	Elim	120721ElimC24HA1b	Factor:ElimC24h | Sample source:Elim Control	RAW_FILE_NAME(Rwaw file name)=120721ElimC24HA1b
SUBJECT_SAMPLE_FACTORS           	Elim	120721ElimC24HA2b	Factor:ElimC24h | Sample source:Elim Control	RAW_FILE_NAME(Rwaw file name)=120721ElimC24HA2b
SUBJECT_SAMPLE_FACTORS           	Elim	120721ElimC24HA3b	Factor:ElimC24h | Sample source:Elim Control	RAW_FILE_NAME(Rwaw file name)=120721ElimC24HA3b
SUBJECT_SAMPLE_FACTORS           	Elim	120721ElimC24HB1b	Factor:ElimC24h | Sample source:Elim Control	RAW_FILE_NAME(Rwaw file name)=120721ElimC24HB1b
SUBJECT_SAMPLE_FACTORS           	Elim	120721ElimC24HB2b	Factor:ElimC24h | Sample source:Elim Control	RAW_FILE_NAME(Rwaw file name)=120721ElimC24HB2b
SUBJECT_SAMPLE_FACTORS           	Elim	120721ElimC24HB3b	Factor:ElimC24h | Sample source:Elim Control	RAW_FILE_NAME(Rwaw file name)=120721ElimC24HB3b
SUBJECT_SAMPLE_FACTORS           	Elim	120721ElimC24HC1b	Factor:ElimC24h | Sample source:Elim Control	RAW_FILE_NAME(Rwaw file name)=120721ElimC24HC1b
SUBJECT_SAMPLE_FACTORS           	Elim	120721ElimC24HC2b	Factor:ElimC24h | Sample source:Elim Control	RAW_FILE_NAME(Rwaw file name)=120721ElimC24HC2b
SUBJECT_SAMPLE_FACTORS           	Elim	120721ElimC24HC3b	Factor:ElimC24h | Sample source:Elim Control	RAW_FILE_NAME(Rwaw file name)=120721ElimC24HC3b
SUBJECT_SAMPLE_FACTORS           	Elim	120721ElimC36HA1b	Factor:ElimC36h | Sample source:Elim Control	RAW_FILE_NAME(Rwaw file name)=120721ElimC36HA1b
SUBJECT_SAMPLE_FACTORS           	Elim	120721ElimC36HA2b	Factor:ElimC36h | Sample source:Elim Control	RAW_FILE_NAME(Rwaw file name)=120721ElimC36HA2b
SUBJECT_SAMPLE_FACTORS           	Elim	120721ElimC36HA3b	Factor:ElimC36h | Sample source:Elim Control	RAW_FILE_NAME(Rwaw file name)=120721ElimC36HA3b
SUBJECT_SAMPLE_FACTORS           	Elim	120721ElimC36HB1b	Factor:ElimC36h | Sample source:Elim Control	RAW_FILE_NAME(Rwaw file name)=120721ElimC36HB1b
SUBJECT_SAMPLE_FACTORS           	Elim	120721ElimC36HB2b	Factor:ElimC36h | Sample source:Elim Control	RAW_FILE_NAME(Rwaw file name)=120721ElimC36HB2b
SUBJECT_SAMPLE_FACTORS           	Elim	120721ElimC36HB3b	Factor:ElimC36h | Sample source:Elim Control	RAW_FILE_NAME(Rwaw file name)=120721ElimC36HB3b
SUBJECT_SAMPLE_FACTORS           	Elim	120721ElimC36HC1b	Factor:ElimC36h | Sample source:Elim Control	RAW_FILE_NAME(Rwaw file name)=120721ElimC36HC1b
SUBJECT_SAMPLE_FACTORS           	Elim	120721ElimC36HC2b	Factor:ElimC36h | Sample source:Elim Control	RAW_FILE_NAME(Rwaw file name)=120721ElimC36HC2b
SUBJECT_SAMPLE_FACTORS           	Elim	120721ElimC36HC3b	Factor:ElimC36h | Sample source:Elim Control	RAW_FILE_NAME(Rwaw file name)=120721ElimC36HC3b
SUBJECT_SAMPLE_FACTORS           	Elim	120721ElimDCAA12HA1b	Factor:ElimDCAA12h | Sample source:Elim DCAA	RAW_FILE_NAME(Rwaw file name)=120721ElimDCAA12HA1b
SUBJECT_SAMPLE_FACTORS           	Elim	120721ElimDCAA12HA2b	Factor:ElimDCAA12h | Sample source:Elim DCAA	RAW_FILE_NAME(Rwaw file name)=120721ElimDCAA12HA2b
SUBJECT_SAMPLE_FACTORS           	Elim	120721ElimDCAA12HA3b	Factor:ElimDCAA12h | Sample source:Elim DCAA	RAW_FILE_NAME(Rwaw file name)=120721ElimDCAA12HA3b
SUBJECT_SAMPLE_FACTORS           	Elim	120721ElimDCAA12HB1b	Factor:ElimDCAA12h | Sample source:Elim DCAA	RAW_FILE_NAME(Rwaw file name)=120721ElimDCAA12HB1b
SUBJECT_SAMPLE_FACTORS           	Elim	120721ElimDCAA12HB2b	Factor:ElimDCAA12h | Sample source:Elim DCAA	RAW_FILE_NAME(Rwaw file name)=120721ElimDCAA12HB2b
SUBJECT_SAMPLE_FACTORS           	Elim	120721ElimDCAA12HB3b	Factor:ElimDCAA12h | Sample source:Elim DCAA	RAW_FILE_NAME(Rwaw file name)=120721ElimDCAA12HB3b
SUBJECT_SAMPLE_FACTORS           	Elim	120721ElimDCAA12HC1b	Factor:ElimDCAA12h | Sample source:Elim DCAA	RAW_FILE_NAME(Rwaw file name)=120721ElimDCAA12HC1b
SUBJECT_SAMPLE_FACTORS           	Elim	120721ElimDCAA12HC2b	Factor:ElimDCAA12h | Sample source:Elim DCAA	RAW_FILE_NAME(Rwaw file name)=120721ElimDCAA12HC2b
SUBJECT_SAMPLE_FACTORS           	Elim	120721ElimDCAA12HC3b	Factor:ElimDCAA12h | Sample source:Elim DCAA	RAW_FILE_NAME(Rwaw file name)=120721ElimDCAA12HC3b
SUBJECT_SAMPLE_FACTORS           	Elim	120721ElimDCAA24HA1b	Factor:ElimDCAA24h | Sample source:Elim DCAA	RAW_FILE_NAME(Rwaw file name)=120721ElimDCAA24HA1b
SUBJECT_SAMPLE_FACTORS           	Elim	120721ElimDCAA24HA2b	Factor:ElimDCAA24h | Sample source:Elim DCAA	RAW_FILE_NAME(Rwaw file name)=120721ElimDCAA24HA2b
SUBJECT_SAMPLE_FACTORS           	Elim	120721ElimDCAA24HA3b	Factor:ElimDCAA24h | Sample source:Elim DCAA	RAW_FILE_NAME(Rwaw file name)=120721ElimDCAA24HA3b
SUBJECT_SAMPLE_FACTORS           	Elim	120721ElimDCAA24HB1b	Factor:ElimDCAA24h | Sample source:Elim DCAA	RAW_FILE_NAME(Rwaw file name)=120721ElimDCAA24HB1b
SUBJECT_SAMPLE_FACTORS           	Elim	120721ElimDCAA24HB2b	Factor:ElimDCAA24h | Sample source:Elim DCAA	RAW_FILE_NAME(Rwaw file name)=120721ElimDCAA24HB2b
SUBJECT_SAMPLE_FACTORS           	Elim	120721ElimDCAA24HB3b	Factor:ElimDCAA24h | Sample source:Elim DCAA	RAW_FILE_NAME(Rwaw file name)=120721ElimDCAA24HB3b
SUBJECT_SAMPLE_FACTORS           	Elim	120721ElimDCAA24HC1b	Factor:ElimDCAA24h | Sample source:Elim DCAA	RAW_FILE_NAME(Rwaw file name)=120721ElimDCAA24HC1b
SUBJECT_SAMPLE_FACTORS           	Elim	120721ElimDCAA24HC2b	Factor:ElimDCAA24h | Sample source:Elim DCAA	RAW_FILE_NAME(Rwaw file name)=120721ElimDCAA24HC2b
SUBJECT_SAMPLE_FACTORS           	Elim	120721ElimDCAA24HC3b	Factor:ElimDCAA24h | Sample source:Elim DCAA	RAW_FILE_NAME(Rwaw file name)=120721ElimDCAA24HC3b
SUBJECT_SAMPLE_FACTORS           	Elim	120721ElimDCAA36HA1b	Factor:ElimDCAA36h | Sample source:Elim DCAA	RAW_FILE_NAME(Rwaw file name)=120721ElimDCAA36HA1b
SUBJECT_SAMPLE_FACTORS           	Elim	120721ElimDCAA36HA2b	Factor:ElimDCAA36h | Sample source:Elim DCAA	RAW_FILE_NAME(Rwaw file name)=120721ElimDCAA36HA2b
SUBJECT_SAMPLE_FACTORS           	Elim	120721ElimDCAA36HA3b	Factor:ElimDCAA36h | Sample source:Elim DCAA	RAW_FILE_NAME(Rwaw file name)=120721ElimDCAA36HA3b
SUBJECT_SAMPLE_FACTORS           	Elim	120721ElimDCAA36HB1b	Factor:ElimDCAA36h | Sample source:Elim DCAA	RAW_FILE_NAME(Rwaw file name)=120721ElimDCAA36HB1b
SUBJECT_SAMPLE_FACTORS           	Elim	120721ElimDCAA36HB2b	Factor:ElimDCAA36h | Sample source:Elim DCAA	RAW_FILE_NAME(Rwaw file name)=120721ElimDCAA36HB2b
SUBJECT_SAMPLE_FACTORS           	Elim	120721ElimDCAA36HB3b	Factor:ElimDCAA36h | Sample source:Elim DCAA	RAW_FILE_NAME(Rwaw file name)=120721ElimDCAA36HB3b
SUBJECT_SAMPLE_FACTORS           	Elim	120721ElimDCAA36HC1b	Factor:ElimDCAA36h | Sample source:Elim DCAA	RAW_FILE_NAME(Rwaw file name)=120721ElimDCAA36HC1b
SUBJECT_SAMPLE_FACTORS           	Elim	120721ElimDCAA36HC2b	Factor:ElimDCAA36h | Sample source:Elim DCAA	RAW_FILE_NAME(Rwaw file name)=120721ElimDCAA36HC2b
SUBJECT_SAMPLE_FACTORS           	Elim	120721ElimDCAA36HC3b	Factor:ElimDCAA36h | Sample source:Elim DCAA	RAW_FILE_NAME(Rwaw file name)=120721ElimDCAA36HC3b
SUBJECT_SAMPLE_FACTORS           	Elim	120721QCAll1b	Factor:QCAll | Sample source:QCAll	RAW_FILE_NAME(Rwaw file name)=120721QCAll1b
SUBJECT_SAMPLE_FACTORS           	Elim	120721QCAll2b	Factor:QCAll | Sample source:QCAll	RAW_FILE_NAME(Rwaw file name)=120721QCAll2b
SUBJECT_SAMPLE_FACTORS           	Elim	120721QCAll3b	Factor:QCAll | Sample source:QCAll	RAW_FILE_NAME(Rwaw file name)=120721QCAll3b
SUBJECT_SAMPLE_FACTORS           	Elim	120721QCAll4b	Factor:QCAll | Sample source:QCAll	RAW_FILE_NAME(Rwaw file name)=120721QCAll4b
SUBJECT_SAMPLE_FACTORS           	Elim	120721QCAll5b	Factor:QCAll | Sample source:QCAll	RAW_FILE_NAME(Rwaw file name)=120721QCAll5b
SUBJECT_SAMPLE_FACTORS           	Elim	120721QCAll6b	Factor:QCAll | Sample source:QCAll	RAW_FILE_NAME(Rwaw file name)=120721QCAll6b
SUBJECT_SAMPLE_FACTORS           	Elim	120721QCAll7b	Factor:QCAll | Sample source:QCAll	RAW_FILE_NAME(Rwaw file name)=120721QCAll7b
#COLLECTION
CO:COLLECTION_SUMMARY            	Seeds from the barley cultivar ‘Elim’ were provided by the South African
CO:COLLECTION_SUMMARY            	Barley Breeding Institute (SABBI, Bredasdorp, Western Cape, South Africa). They
CO:COLLECTION_SUMMARY            	were surfaced-sterilised with 70% ethanol and soaked in sterile water for 2 h
CO:COLLECTION_SUMMARY            	prior to cultivation in soil (Germination mix, Culterra, Muldersdrift, South
CO:COLLECTION_SUMMARY            	Africa) pasteurised at 70 °C. An average of 40 seeds were planted in each pot
CO:COLLECTION_SUMMARY            	(three pots per condition or three biological replicate) measuring 8 cm depth
CO:COLLECTION_SUMMARY            	and 12 cm in diameter. The watering of the plants was performed twice a week
CO:COLLECTION_SUMMARY            	with water and a solution containing a water-soluble chemical fertiliser
CO:COLLECTION_SUMMARY            	(Multisol ‘N’, Culterra, Muldersdrift, South Africa). Seedlings were kept in
CO:COLLECTION_SUMMARY            	a regulated growth environment with a 12-hour light-dark cycle at 22 to 27°C
CO:COLLECTION_SUMMARY            	until 16 d post-emergence or 21 d after planting, corresponding to physiological
CO:COLLECTION_SUMMARY            	stage 13 according to the Zadocks growth and development scale (Zadoks et al.
CO:COLLECTION_SUMMARY            	1974). The priming inducer 3,5-dichloroanthranilic acid (3,5-DCAA) was purchased
CO:COLLECTION_SUMMARY            	from Merck-Sigma-Aldrich, (Johannesburg, South Africa). 3,5-DCAA was dissolved
CO:COLLECTION_SUMMARY            	in dimethylsulphoxide (DMSO, 1 μL.mL-1; BDH Chemicals, UK) and mixed with 0.05%
CO:COLLECTION_SUMMARY            	wetting agent (Effekto, Pretoria, South Africa) in distilled water to obtain the
CO:COLLECTION_SUMMARY            	desired concentrations. Approximately 6 mL (40 sprays) of 200 μM 3,5-DCAA was
CO:COLLECTION_SUMMARY            	applied on the leaf tissue of the seedlings while the controls received only the
CO:COLLECTION_SUMMARY            	DMSO solution. The leaf material was harvested at 12, 24 and 36 h post-treatment
CO:COLLECTION_SUMMARY            	and snap-frozen in liquid nitrogen to quench metabolic activity. Samples were
CO:COLLECTION_SUMMARY            	stored in -80 °C for later use. Metabolites were extracted as previously
CO:COLLECTION_SUMMARY            	described (Hamany Djande et al., 2023b). Briefly, the leaf tissue was ground
CO:COLLECTION_SUMMARY            	with liquid nitrogen, then 80% methanol was added and the mixture was
CO:COLLECTION_SUMMARY            	homogenized. Subsequently, all hydromethanolic samples were centrifuged, and the
CO:COLLECTION_SUMMARY            	supernatant was concentrated and further evaporated to complete dryness. The
CO:COLLECTION_SUMMARY            	dried extracts were dissolved in 50% methanol, filtered, and prepared for LC-MS
CO:COLLECTION_SUMMARY            	analysis.
CO:SAMPLE_TYPE                   	Plant shoot tissue
#TREATMENT
TR:TREATMENT_SUMMARY             	3,5-DCAA was dissolved in dimethylsulphoxide (DMSO, 1 μL.mL-1; BDH Chemicals,
TR:TREATMENT_SUMMARY             	UK) and mixed with 0.05% wetting agent (Effekto, Pretoria, South Africa) in
TR:TREATMENT_SUMMARY             	distilled water to obtain the desired concentrations. Approximately 6 mL (40
TR:TREATMENT_SUMMARY             	sprays) of 200 μM 3,5-DCAA was applied on the leaf tissue of the seedlings
TR:TREATMENT_SUMMARY             	while the controls received only the DMSO solution. The leaf material was
TR:TREATMENT_SUMMARY             	harvested at 12, 24 and 36 h post-treatment and snap-frozen in liquid nitrogen
TR:TREATMENT_SUMMARY             	to quench metabolic activity. Samples were stored in -80 °C for later use.
TR:TREATMENT                     	synthetic inducer
TR:TREATMENT_COMPOUND            	3,5-DCAA
TR:TREATMENT_DOSE                	Approximately 6 mL (40 sprays) of 200 μM 3,5-DCAA
TR:TREATMENT_DOSEVOLUME          	6 mL
TR:PLANT_WATERING_REGIME         	Twice a week
TR:PLANT_GROWTH_STAGE            	stage 13 according to the Zadocks growth and development scale (Zadoks et al.
TR:PLANT_GROWTH_STAGE            	1974)
TR:PLANT_STORAGE                 	-80
#SAMPLEPREP
SP:SAMPLEPREP_SUMMARY            	Metabolites were extracted as previously described (Hamany Djande et al.,
SP:SAMPLEPREP_SUMMARY            	2023b). Briefly, the leaf tissue was ground with liquid nitrogen, then 80%
SP:SAMPLEPREP_SUMMARY            	methanol was added and the mixture was homogenized. Subsequently, all
SP:SAMPLEPREP_SUMMARY            	hydromethanolic samples were centrifuged, and the supernatant was concentrated
SP:SAMPLEPREP_SUMMARY            	and further evaporated to complete dryness. The dried extracts were dissolved in
SP:SAMPLEPREP_SUMMARY            	50% methanol, filtered, and prepared for LC-MS analysis.
#CHROMATOGRAPHY
CH:CHROMATOGRAPHY_SUMMARY        	The analysis of the aqueous methanol extracts was performed with a Waters
CH:CHROMATOGRAPHY_SUMMARY        	Acquity UHPLC coupled to a Waters SYNAPT G1 QTOF (quadrupole time-of-flight)
CH:CHROMATOGRAPHY_SUMMARY        	high definition mass spectrometer system (Waters Corporation, Milford, MA, USA).
CH:CHROMATOGRAPHY_SUMMARY        	The HSS T3 C18 column (150 mm x 2.1 mm x 1.8 µm, Waters Corporation) was
CH:CHROMATOGRAPHY_SUMMARY        	thermostatted at 60 °C and used for the reverse phase chromatographic
CH:CHROMATOGRAPHY_SUMMARY        	separation of extracts. The mobile phase consisted of mass spectrometry grade
CH:CHROMATOGRAPHY_SUMMARY        	water and acetonitrile (Romil, SpS, Cambridge, UK) and formic acid
CH:CHROMATOGRAPHY_SUMMARY        	(Sigma-Aldrich, Merck, Johannesburg, South Africa). Eluents A (water), and B
CH:CHROMATOGRAPHY_SUMMARY        	(acetonitrile), both containing 0.1% formic acid were used for the concave
CH:CHROMATOGRAPHY_SUMMARY        	gradient elution running at a flow rate of 0.4 mL min−1. The elution commenced
CH:CHROMATOGRAPHY_SUMMARY        	with 5% B for the first min, and gradually increased to 95% B over 24 min. The
CH:CHROMATOGRAPHY_SUMMARY        	chromatographic conditions were then adjusted to 10% A and 90% B, for 10 s,
CH:CHROMATOGRAPHY_SUMMARY        	followed by 5% A and 95% B for 1 min 50 s before restoration to the initial
CH:CHROMATOGRAPHY_SUMMARY        	conditions for column equilibration for 2 min. The injection volume was 2 µL
CH:CHROMATOGRAPHY_SUMMARY        	and the total run time was 30 min. To account for analytical variability and to
CH:CHROMATOGRAPHY_SUMMARY        	prevent measurement bias, each sample was analysed in triplicate. The sample
CH:CHROMATOGRAPHY_SUMMARY        	order was randomised and blanks consisting of 50% methanol were injected to
CH:CHROMATOGRAPHY_SUMMARY        	monitor the background noise, possible sample carry-over and solvent
CH:CHROMATOGRAPHY_SUMMARY        	contamination. The stability of the LC-MS system was monitored by inserting
CH:CHROMATOGRAPHY_SUMMARY        	quality control (QC) samples in the batches. Data acquisition involved three
CH:CHROMATOGRAPHY_SUMMARY        	independent biological replicates, with each replicate analysed in triplicate,
CH:CHROMATOGRAPHY_SUMMARY        	resulting in a total sample size of n = 9.
CH:CHROMATOGRAPHY_TYPE           	Reversed phase
CH:INSTRUMENT_NAME               	Waters Acquity
CH:COLUMN_NAME                   	Waters HSS T3 C18 (150 x 2.1mm x 1.8um)
CH:SOLVENT_A                     	100% water; 0.1% formic acid
CH:SOLVENT_B                     	100% acetonitrile; 0.1% formic acid
CH:FLOW_GRADIENT                 	The elution commenced with 5% B for the first min, and gradually increased to
CH:FLOW_GRADIENT                 	95% B over 24 min. The chromatographic conditions were then adjusted to 10% A
CH:FLOW_GRADIENT                 	and 90% B, for 10 s, followed by 5% A and 95% B for 1 min 50 s before
CH:FLOW_GRADIENT                 	restoration to the initial conditions for column equilibration for 2 min. The
CH:FLOW_GRADIENT                 	injection volume was 2 µL and the total run time was 30 min
CH:FLOW_RATE                     	0.4 mL/min
CH:COLUMN_TEMPERATURE            	N/A
#ANALYSIS
AN:ANALYSIS_TYPE                 	MS
#MS
MS:INSTRUMENT_NAME               	Waters SYNAPT G1 QTOF
MS:INSTRUMENT_TYPE               	QTOF
MS:MS_TYPE                       	ESI
MS:ION_MODE                      	NEGATIVE
MS:MS_COMMENTS                   	The high resolution, accurate mass TOF-MS analyser was used in a V-optics mode,
MS:MS_COMMENTS                   	with the centroid spectral data acquired in negative electrospray ionisation
MS:MS_COMMENTS                   	(ESI) modes. Operating parameters included masses ranging from 50 to 1200 Da and
MS:MS_COMMENTS                   	a scan time of 0.1 s; the capillary voltage set at 2.5 kV; sampling and
MS:MS_COMMENTS                   	extraction cone voltages at 40 V and 4.0 V, respectively. The desolvation and
MS:MS_COMMENTS                   	cone gas flows were set at 550 L h−1 and 50 L h−1, respectively, with
MS:MS_COMMENTS                   	nitrogen used as the nebulisation gas at a flow rate of 700 L h−1. A
MS:MS_COMMENTS                   	desolvation temperature of 450 °C and a fixed source temperature of 120 °C
MS:MS_COMMENTS                   	were used. Leucine encephalin ([M-H]− = 554.2615 and [M + H]+ = 556.2766) at a
MS:MS_COMMENTS                   	concentration of 50 pg mL−1, served as the reference mass calibrant, and was
MS:MS_COMMENTS                   	sampled every 15 sec to generate an average intensity of 350 counts per scan.
MS:MS_COMMENTS                   	This reference helped the processing software (MassLynx XSTM 4.1, Waters
MS:MS_COMMENTS                   	Corporation, Milford, MA, USA) to perform automatic correction of slight
MS:MS_COMMENTS                   	centroid mass deviations observed in the samples, ensuring precise mass
MS:MS_COMMENTS                   	measurements with typical mass accuracy ranging from 1 to 3 mDa. Both intact and
MS:MS_COMMENTS                   	fragmented data were aquired using an MSE method with collision energies ranging
MS:MS_COMMENTS                   	from 10 to 40 eV. The fragmentation data were employed for subsequent metabolite
MS:MS_COMMENTS                   	structural elucidation and annotation.
MS:MS_RESULTS_FILE               	ST003399_AN005579_Results.txt	UNITS:m/z_rt	Has m/z:Yes	Has RT:Yes	RT units:Minutes
#END