#METABOLOMICS WORKBENCH MicrometaboliteNR_20211128_084635 DATATRACK_ID:2951 STUDY_ID:ST002021 ANALYSIS_ID:AN003291 PROJECT_ID:PR001283
VERSION             	1
CREATED_ON             	November 29, 2021, 7:54 am
#PROJECT
PR:PROJECT_TITLE                 	MICROMETABOLITE
PR:PROJECT_SUMMARY               	The overall objective of MICROMETABOLITE is to explore interactions between
PR:PROJECT_SUMMARY               	plants and microorganisms involved in the production of secondary metabolites
PR:PROJECT_SUMMARY               	(SM) for introducing novel ingredients in pharmaceutical and cosmeceutical
PR:PROJECT_SUMMARY               	industry. Effects of microorganisms on the plant metabolome and the biosynthesis
PR:PROJECT_SUMMARY               	of bioactive SM will be studied in the Boraginaceae plant family, aimed at
PR:PROJECT_SUMMARY               	optimising plant cultivation and alkannins/shikonins (A/S) production.
PR:PROJECT_SUMMARY               	Microorganisms will be integrated in plant production systems, and protocols
PR:PROJECT_SUMMARY               	needed for efficient implementation in industry will be elaborated. Thereby a
PR:PROJECT_SUMMARY               	platform will be established that will support long-term interactions between
PR:PROJECT_SUMMARY               	academia and industry.
PR:INSTITUTE                     	Aristotle University of Thessaloniki, MICROMETABOLITE project
PR:DEPARTMENT                    	School of Chemical Engineering
PR:LAST_NAME                     	Rodic
PR:FIRST_NAME                    	Nebojsa
PR:ADDRESS                       	Stepe Stepanovica 5, Conoplja, Vojvodina, 25210, Yugoslavia
PR:EMAIL                         	nebojsa.rodic@hotmail.com
PR:PHONE                         	+381648766400
PR:FUNDING_SOURCE                	This research was supported by the European Union's Horizon 2020 research and
PR:FUNDING_SOURCE                	innovation programme under the Marie Skłodowska-Curie grant agreement No 721635
#STUDY
ST:STUDY_TITLE                   	An integrated-omics approach reveals specific bacterial and fungal taxa
ST:STUDY_TITLE                   	associated with roots of Alkanna tinctoria L. Tausch correlating with
ST:STUDY_TITLE                   	medicinally relevant alkannin derivatives and other secondary metabolites
ST:STUDY_SUMMARY                 	Plants are naturally associated with diverse microbial communities, which play
ST:STUDY_SUMMARY                 	significant roles in plant performance, such as growth promotion or fending off
ST:STUDY_SUMMARY                 	pathogens. The roots of Alkanna tinctoria L. are rich in naphthoquinones,
ST:STUDY_SUMMARY                 	particularly the medicinally used chiral compounds alkannin, shikonin and their
ST:STUDY_SUMMARY                 	derivatives. Former studies already have shown that microorganisms may modulate
ST:STUDY_SUMMARY                 	plant metabolism. To further investigate the potential interaction between A.
ST:STUDY_SUMMARY                 	tinctoria and associated microorganisms we performed a greenhouse experiment, in
ST:STUDY_SUMMARY                 	which A. tinctoria plants were grown in the presence of three distinct soil
ST:STUDY_SUMMARY                 	microbiomes. At four defined plant developmental stages we made an in-depth
ST:STUDY_SUMMARY                 	assessment of bacterial and fungal root-associated microbiomes as well as all
ST:STUDY_SUMMARY                 	primary and secondary metabolites. Our results showed that the plant
ST:STUDY_SUMMARY                 	developmental stage was the most important driver influencing the plant
ST:STUDY_SUMMARY                 	metabolite content, revealing peak contents of alkannin/shikonin at the fruiting
ST:STUDY_SUMMARY                 	stage. In contrast, the soil microbiome had the biggest impact on the plant root
ST:STUDY_SUMMARY                 	microbiome. Correlation analyses performed on the measured metabolite content
ST:STUDY_SUMMARY                 	and the abundance of individual bacterial and fungal taxa suggested a dynamic,
ST:STUDY_SUMMARY                 	at times positive or negative relationship between root-associated
ST:STUDY_SUMMARY                 	microorganisms and root metabolism. In particular, the bacterial
ST:STUDY_SUMMARY                 	Allorhizobium-Neorhizobium-Pararhizobium-Rhizobium group and the fungal species
ST:STUDY_SUMMARY                 	Penicillium jensenii were found to be positively correlated with higher content
ST:STUDY_SUMMARY                 	of alkannins.
ST:INSTITUTE                     	Aristotle University of Thessaloniki, MICROMETABOLITE project
ST:DEPARTMENT                    	School of Chemical Engineering
ST:LAST_NAME                     	Rodic
ST:FIRST_NAME                    	Nebojsa
ST:ADDRESS                       	Stepe Stepanovica 5, Conoplja, Vojvodina, 25210, Yugoslavia
ST:EMAIL                         	nebojsa.rodic@hotmail.com
ST:PHONE                         	+381648766400
#SUBJECT
SU:SUBJECT_TYPE                  	Plant
SU:SUBJECT_SPECIES               	Alkanna tinctoria
#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           	-	I.1	developmental stage:growth | soil type:Greek A	dilution factor=1; RAW_FILE_NAME=1-3_RA_1.raw
SUBJECT_SAMPLE_FACTORS           	-	I.2	developmental stage:growth | soil type:Greek A	dilution factor=1; RAW_FILE_NAME=1-3_RA_2.raw
SUBJECT_SAMPLE_FACTORS           	-	I.3	developmental stage:growth | soil type:Greek A	dilution factor=1; RAW_FILE_NAME=1-3_RA_3.raw
SUBJECT_SAMPLE_FACTORS           	-	I.4	developmental stage:growth | soil type:Greek A	dilution factor=1; RAW_FILE_NAME=1-3_RA_4.raw
SUBJECT_SAMPLE_FACTORS           	-	I.5	developmental stage:growth | soil type:Greek A	dilution factor=1; RAW_FILE_NAME=1-3_RA_5.raw
SUBJECT_SAMPLE_FACTORS           	-	I.6	developmental stage:growth | soil type:Greek A	dilution factor=1; RAW_FILE_NAME=1-3_RA_6.raw
SUBJECT_SAMPLE_FACTORS           	-	I.7	developmental stage:growth | soil type:Greek B	dilution factor=1; RAW_FILE_NAME=1-3_RA_7.raw
SUBJECT_SAMPLE_FACTORS           	-	I.8	developmental stage:growth | soil type:Greek B	dilution factor=1; RAW_FILE_NAME=1-3_RA_8.raw
SUBJECT_SAMPLE_FACTORS           	-	I.9	developmental stage:growth | soil type:Greek B	dilution factor=1; RAW_FILE_NAME=1-3_RA_9.raw
SUBJECT_SAMPLE_FACTORS           	-	I.10	developmental stage:growth | soil type:Greek B	dilution factor=1; RAW_FILE_NAME=1-3_RA_10.raw
SUBJECT_SAMPLE_FACTORS           	-	I.11	developmental stage:growth | soil type:Greek B	dilution factor=1; RAW_FILE_NAME=1-3_RA_11.raw
SUBJECT_SAMPLE_FACTORS           	-	I.12	developmental stage:growth | soil type:Greek B	dilution factor=1; RAW_FILE_NAME=1-3_RA_12.raw
SUBJECT_SAMPLE_FACTORS           	-	I.13	developmental stage:growth | soil type:Austrian	dilution factor=1; RAW_FILE_NAME=1-3_RA_13.raw
SUBJECT_SAMPLE_FACTORS           	-	I.14	developmental stage:growth | soil type:Austrian	dilution factor=1; RAW_FILE_NAME=1-3_RA_14.raw
SUBJECT_SAMPLE_FACTORS           	-	I.15	developmental stage:growth | soil type:Austrian	dilution factor=1; RAW_FILE_NAME=1-3_RA_15.raw
SUBJECT_SAMPLE_FACTORS           	-	I.16	developmental stage:growth | soil type:Austrian	dilution factor=1; RAW_FILE_NAME=1-3_RA_16.raw
SUBJECT_SAMPLE_FACTORS           	-	I.17	developmental stage:growth | soil type:Austrian	dilution factor=1; RAW_FILE_NAME=1-3_RA_17.raw
SUBJECT_SAMPLE_FACTORS           	-	I.18	developmental stage:growth | soil type:Austrian	dilution factor=1; RAW_FILE_NAME=1-3_RA_18.raw
SUBJECT_SAMPLE_FACTORS           	-	II.1	developmental stage:blooming | soil type:Greek A	dilution factor=1; RAW_FILE_NAME=15-3_RA_1.raw
SUBJECT_SAMPLE_FACTORS           	-	II.2	developmental stage:blooming | soil type:Greek A	dilution factor=1; RAW_FILE_NAME=15-3_RA_2.raw
SUBJECT_SAMPLE_FACTORS           	-	II.3	developmental stage:blooming | soil type:Greek A	dilution factor=1; RAW_FILE_NAME=15-3_RA_3.raw
SUBJECT_SAMPLE_FACTORS           	-	II.4	developmental stage:blooming | soil type:Greek A	dilution factor=1; RAW_FILE_NAME=15-3_RA_4.raw
SUBJECT_SAMPLE_FACTORS           	-	II.5	developmental stage:blooming | soil type:Greek A	dilution factor=1; RAW_FILE_NAME=15-3_RA_5.raw
SUBJECT_SAMPLE_FACTORS           	-	II.6	developmental stage:blooming | soil type:Greek A	dilution factor=1; RAW_FILE_NAME=15-3_RA_6.raw
SUBJECT_SAMPLE_FACTORS           	-	II.7	developmental stage:blooming | soil type:Greek B	dilution factor=1; RAW_FILE_NAME=15-3_RA_7.raw
SUBJECT_SAMPLE_FACTORS           	-	II.8	developmental stage:blooming | soil type:Greek B	dilution factor=1; RAW_FILE_NAME=15-3_RA_8.raw
SUBJECT_SAMPLE_FACTORS           	-	II.9	developmental stage:blooming | soil type:Greek B	dilution factor=1; RAW_FILE_NAME=15-3_RA_9.raw
SUBJECT_SAMPLE_FACTORS           	-	II.10	developmental stage:blooming | soil type:Greek B	dilution factor=1; RAW_FILE_NAME=15-3_RA_10.raw
SUBJECT_SAMPLE_FACTORS           	-	II.11	developmental stage:blooming | soil type:Greek B	dilution factor=1; RAW_FILE_NAME=15-3_RA_11.raw
SUBJECT_SAMPLE_FACTORS           	-	II.12	developmental stage:blooming | soil type:Greek B	dilution factor=1; RAW_FILE_NAME=15-3_RA_12.raw
SUBJECT_SAMPLE_FACTORS           	-	II.13	developmental stage:blooming | soil type:Austrian	dilution factor=1; RAW_FILE_NAME=15-3_RA_13.raw
SUBJECT_SAMPLE_FACTORS           	-	II.14	developmental stage:blooming | soil type:Austrian	dilution factor=1; RAW_FILE_NAME=15-3_RA_14.raw
SUBJECT_SAMPLE_FACTORS           	-	II.15	developmental stage:blooming | soil type:Austrian	dilution factor=1; RAW_FILE_NAME=15-3_RA_15.raw
SUBJECT_SAMPLE_FACTORS           	-	II.16	developmental stage:blooming | soil type:Austrian	dilution factor=1; RAW_FILE_NAME=15-3_RA_16.raw
SUBJECT_SAMPLE_FACTORS           	-	II.17	developmental stage:blooming | soil type:Austrian	dilution factor=1; RAW_FILE_NAME=15-3_RA_17.raw
SUBJECT_SAMPLE_FACTORS           	-	II.18	developmental stage:blooming | soil type:Austrian	dilution factor=1; RAW_FILE_NAME=15-3_RA_18.raw
SUBJECT_SAMPLE_FACTORS           	-	III.1	developmental stage:fruiting | soil type:Greek A	dilution factor=1; RAW_FILE_NAME=12-4_RA_1.raw
SUBJECT_SAMPLE_FACTORS           	-	III.2	developmental stage:fruiting | soil type:Greek A	dilution factor=1; RAW_FILE_NAME=12-4_RA_2.raw
SUBJECT_SAMPLE_FACTORS           	-	III.3	developmental stage:fruiting | soil type:Greek A	dilution factor=1; RAW_FILE_NAME=12-4_RA_3.raw
SUBJECT_SAMPLE_FACTORS           	-	III.4	developmental stage:fruiting | soil type:Greek A	dilution factor=3; RAW_FILE_NAME=12-4_RA_4D.raw
SUBJECT_SAMPLE_FACTORS           	-	III.5	developmental stage:fruiting | soil type:Greek A	dilution factor=1; RAW_FILE_NAME=12-4_RA_5.raw
SUBJECT_SAMPLE_FACTORS           	-	III.6	developmental stage:fruiting | soil type:Greek A	dilution factor=3; RAW_FILE_NAME=12-4_RA_6D.raw
SUBJECT_SAMPLE_FACTORS           	-	III.7	developmental stage:fruiting | soil type:Greek B	dilution factor=1; RAW_FILE_NAME=12-4_RA_7.raw
SUBJECT_SAMPLE_FACTORS           	-	III.8	developmental stage:fruiting | soil type:Greek B	dilution factor=3; RAW_FILE_NAME=12-4_RA_8D.raw
SUBJECT_SAMPLE_FACTORS           	-	III.9	developmental stage:fruiting | soil type:Greek B	dilution factor=3; RAW_FILE_NAME=12-4_RA_9D.raw
SUBJECT_SAMPLE_FACTORS           	-	III.10	developmental stage:fruiting | soil type:Greek B	dilution factor=1; RAW_FILE_NAME=12-4_RA_10.raw
SUBJECT_SAMPLE_FACTORS           	-	III.11	developmental stage:fruiting | soil type:Greek B	dilution factor=1; RAW_FILE_NAME=12-4_RA_11.raw
SUBJECT_SAMPLE_FACTORS           	-	III.12	developmental stage:fruiting | soil type:Greek B	dilution factor=3; RAW_FILE_NAME=12-4_RA_12D.raw
SUBJECT_SAMPLE_FACTORS           	-	III.13	developmental stage:fruiting | soil type:Austrian	dilution factor=1; RAW_FILE_NAME=12-4_RA_13.raw
SUBJECT_SAMPLE_FACTORS           	-	III.14	developmental stage:fruiting | soil type:Austrian	dilution factor=1; RAW_FILE_NAME=12-4_RA_14.raw
SUBJECT_SAMPLE_FACTORS           	-	III.15	developmental stage:fruiting | soil type:Austrian	dilution factor=3; RAW_FILE_NAME=12-4_RA_15D.raw
SUBJECT_SAMPLE_FACTORS           	-	III.16	developmental stage:fruiting | soil type:Austrian	dilution factor=3; RAW_FILE_NAME=12-4_RA_16D.raw
SUBJECT_SAMPLE_FACTORS           	-	III.17	developmental stage:fruiting | soil type:Austrian	dilution factor=1; RAW_FILE_NAME=12-4_RA_17.raw
SUBJECT_SAMPLE_FACTORS           	-	III.18	developmental stage:fruiting | soil type:Austrian	dilution factor=3; RAW_FILE_NAME=12-4_RA_18D.raw
SUBJECT_SAMPLE_FACTORS           	-	QC_UCL_AT_01	developmental stage:QC | soil type:NA	dilution factor=1; RAW_FILE_NAME=QC_UCL_AT.raw
SUBJECT_SAMPLE_FACTORS           	-	QC_UCL_AT_02	developmental stage:QC | soil type:NA	dilution factor=1; RAW_FILE_NAME=QC_UCL_AT_180730210457.raw
SUBJECT_SAMPLE_FACTORS           	-	QC_UCL_AT_03	developmental stage:QC | soil type:NA	dilution factor=1; RAW_FILE_NAME=QC_UCL_AT_180731013653.raw
SUBJECT_SAMPLE_FACTORS           	-	QC_UCL_AT_04	developmental stage:QC | soil type:NA	dilution factor=1; RAW_FILE_NAME=QC_UCL_AT_04.raw
SUBJECT_SAMPLE_FACTORS           	-	QC_UCL_AT_05	developmental stage:QC | soil type:NA	dilution factor=1; RAW_FILE_NAME=QC_UCL_AT_05.raw
SUBJECT_SAMPLE_FACTORS           	-	QC_UCL_AT_06	developmental stage:QC | soil type:NA	dilution factor=1; RAW_FILE_NAME=QC_UCL_AT_06.raw
SUBJECT_SAMPLE_FACTORS           	-	QC_UCL_AT_07	developmental stage:QC | soil type:NA	dilution factor=1; RAW_FILE_NAME=QC_UCL_AT_07.raw
SUBJECT_SAMPLE_FACTORS           	-	QC_UCL_AT_08	developmental stage:QC | soil type:NA	dilution factor=1; RAW_FILE_NAME=QC_UCL_AT_08.raw
SUBJECT_SAMPLE_FACTORS           	-	QC_UCL_AT_09	developmental stage:QC | soil type:NA	dilution factor=1; RAW_FILE_NAME=QC_UCL_AT_09.raw
#COLLECTION
CO:COLLECTION_SUMMARY            	Alkanna tinctoria plants were provided as rooted acclimatized individuals,
CO:COLLECTION_SUMMARY            	originally collected and identified from natural populations
CO:SAMPLE_TYPE                   	Plant
#TREATMENT
TR:TREATMENT_SUMMARY             	Plants were produced by micropropagation from several mother plants by the
TR:TREATMENT_SUMMARY             	Hellenic Agricultural Organization (HAO, Thessaloniki, Greece). The plants were
TR:TREATMENT_SUMMARY             	transferred to 5 L pots containing 4.5 L of sterilized (121° C for 15 min) peat
TR:TREATMENT_SUMMARY             	moss and perlite (volume ratio 2:1), mixed with 200 g field soil collected
TR:TREATMENT_SUMMARY             	either in Austria or in Greece. Thus, all plants were grown in a substrate with
TR:TREATMENT_SUMMARY             	highly similar chemical and physical characteristics, but hosting those
TR:TREATMENT_SUMMARY             	microbial communities prevailing in these three distinct soils. Plants were
TR:TREATMENT_SUMMARY             	grown in the greenhouse at 16 h light / 8 h dark photoperiod, 25°C with 50%
TR:TREATMENT_SUMMARY             	relative humidity (RH) and a photosynthetic photon flux density (PPFD) of 96
TR:TREATMENT_SUMMARY             	μmolm^-2 s^-1. Plants were watered twice per week with deionized water and
TR:TREATMENT_SUMMARY             	moved randomly once per week. Plants were harvested at four different defined
TR:TREATMENT_SUMMARY             	developmental stages, the first stage (“vegetative growth”) was defined when
TR:TREATMENT_SUMMARY             	more than 50% of the individuals started to produce new leaves, “blooming”
TR:TREATMENT_SUMMARY             	was the stage when more than 50% of the individuals had flowers, “fruiting”
TR:TREATMENT_SUMMARY             	when more than 50% of the individual plants began to produce fruits.
#SAMPLEPREP
SP:SAMPLEPREP_SUMMARY            	Plant roots were ground to a fine powder using a ball mill (Fritsch Pulverisette
SP:SAMPLEPREP_SUMMARY            	0, Germany). Each powdered sample was weighed (70 mg) into microcentrifuge
SP:SAMPLEPREP_SUMMARY            	tubes, followed by extraction with 3 mL of methanol by ultrasound at 10% power
SP:SAMPLEPREP_SUMMARY            	for 3 h (Bandelin Sonorex Digital 10P, Berlin, Germany) and centrifugation for
SP:SAMPLEPREP_SUMMARY            	10 minutes at 12.500 rpm (Hermle Z 216 MK, Wehingen, Germany). The supernatants
SP:SAMPLEPREP_SUMMARY            	were collected and subjected to UHPLC-HRMS analysis after filtering with 0.22
SP:SAMPLEPREP_SUMMARY            	μm syringe filters.
#CHROMATOGRAPHY
CH:CHROMATOGRAPHY_TYPE           	Reversed phase
CH:INSTRUMENT_NAME               	Thermo Accela AS
CH:COLUMN_NAME                   	Waters Acquity UPLC HSS C18 SB 1.8 μm 2.1 x 100 mm
CH:FLOW_RATE                     	0.3mL/min
CH:COLUMN_TEMPERATURE            	50
#ANALYSIS
AN:ANALYSIS_TYPE                 	MS
#MS
MS:INSTRUMENT_NAME               	Thermo LTQ Discovery Orbitrap
MS:INSTRUMENT_TYPE               	Orbitrap
MS:MS_TYPE                       	ESI
MS:ION_MODE                      	POSITIVE
MS:MS_COMMENTS                   	The MS/MS data were obtained for the six most intense m/z peaks in each full
MS:MS_COMMENTS                   	scan, with the normalized collision energy set to 35 eV. The acquisition and
MS:MS_COMMENTS                   	initial processing of the data were by means of XcaliburTM (Thermo Scientific,
MS:MS_COMMENTS                   	USA) software, while data alignment and feature extraction were performed
MS:MS_COMMENTS                   	utilizing the XCMS Online platform (The Scripps Research Institute, USA). The
MS:MS_COMMENTS                   	batch error correction was done with the help of MetaboAnalyst 5.0.
MS:MS_RESULTS_FILE               	ST002021_AN003291_Results.txt	UNITS:intensity units	Has m/z:Yes	Has RT:Yes	RT units:Minutes
#END